Funding for this work was provided by the University of San Diego, the National Science Foundation Graduate Research Fellowship Program, NIH grant R01-HG007836, and by the Korean Ministry of Science, ICT and Future Planning.
Earlier versions of a several figures were first published in "Hoople, G. D. et al. Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers. Lab on a Chip 17, 2619-2630, doi:10.1039/c7lc00430c (2017)." Lab on a Chip has sanctioned the reuse of figures in this publication.

1. Chemical Solution Preparation
NOTE: The following steps are for preparing chemical solutions required in later steps. These can be made in bulk and stored for several months.
<ol>
	<li>To begin, prepare 50 mL of purified, deionized water by sterilizing in a 254 nm UV crosslinking oven for 15 min (15 mJ/cm2 total exposure) to neutralize any contaminating DNA. Heat to 37 &#176;C for use in following steps.</li>
	<li>Make 10 mL of a 40% total (T) and 3.3% crosslinker (C) (29:1) po...

<ol>
	<li>Mawy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing.">Single-cell sequencing.</a> Nat Methods. 11 (1), 18 (2014).</li><li>Gole, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+polymerase+cloning+and+genome+sequencing+of+single+cells+using+nanoliter+microwells.">Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells.</a> Nat Biotech. 31 (12), 1126-1132 (2013).</li><li>Sasagawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quartz-Seq:+a+highly+reproducible+and+sensitive+single-cell+RNA-Seq+reveals+non-genetic+gene+expression+heterogeneity.">Quartz-Seq: a highly reproducible and sensitive single-cell RNA-Seq reveals non-genetic gene expression heterogeneity.</a> Genome Biol. 14 (4), 31 (2013).</li><li>Ramsk&#246;ld, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+mRNA-Seq+from+single-cell+levels+of+RNA+and+individual+circulating+tumor+cells.">Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.</a> Nat Biotechnol. 30 (8), 777-782 (2012).</li><li>Shapiro, E., Biezuner, T., Linnarsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing-based+technologies+will+revolutionize+whole-organism+science.">Single-cell sequencing-based technologies will revolutionize whole-organism science.</a> Nat Rev Genet. 14 (9), 618-630 (2013).</li><li>Navin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+evolution+inferred+by+single-cell+sequencing.">Tumour evolution inferred by single-cell sequencing.</a> Nature. 472 (7341), 90-94 (2011).</li><li>Hoople, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-seq:+whole-genome+and+transcriptome+sequencing+by+simultaneous+low-input+DNA+and+RNA+library+preparation+using+semi-permeable+hydrogel+barriers.">Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.</a> Lab Chip. 17, 2619-2630 (2017).</li><li>Macaulay, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&amp;T-seq:+parallel+sequencing+of+single-cell+genomes+and+transcriptomes.">G&amp;T-seq: parallel sequencing of single-cell genomes and transcriptomes.</a> Nat Methods. 12 (6), 519-522 (2015).</li><li>Dey, S. S., Kester, L., Spanjaard, B., Bienko, M., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genome+and+transcriptome+sequencing+of+the+same+cell.">Integrated genome and transcriptome sequencing of the same cell.</a> Nat Biotechnol. 33 (3), 285-289 (2015).</li><li>Gopal, A., Zhou, Z. H., Knobler, C. M., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+large+RNA+molecules+in+solution.">Visualizing large RNA molecules in solution.</a> RNA. 18 (2), 284-299 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-PAGE+Gel.">SDS-PAGE Gel.</a> Cold Spring Harb Protoc. 2015 (7), 087908 (2015).</li><li>Lake, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtypes+and+diversity+revealed+by+single-nucleus+RNA+sequencing+of+the+human+brain.">Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain.</a> Science. 352 (6293), (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Qubit 3.0 Fluorometer Manual. MAN0010866. Life Technologies Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/qubit_3_fluorometer_man.pd">https://tools.thermofisher.com/content/sfs/manuals/qubit_3_fluorometer_man.pd</a> (2017)</li><li>Vitak, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+thousands+of+single-cell+genomes+with+combinatorial+indexing.">Sequencing thousands of single-cell genomes with combinatorial indexing.</a> Nat Methods. 14 (3), 302-308 (2017).</li><li>Illumina. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nextera &#174; XT DNA Library Preparation Kit. , (2017).</li><li>Takara Bio USA Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing User Manual. , (2016).</li><li>Kurimoto, K., Yabuta, Y., Ohinata, Y., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+single-cell+cDNA+amplification+to+provide+a+template+for+representative+high-density+oligonucleotide+microarray+analysis.">Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis.</a> Nat Protoc. 2 (3), 739-752 (2007).</li></ol>

There are several critical steps associated with the Gel-seq device fabrication as well as the protocol itself. During fabrication, we recommend starting with the prescribed layer thicknesses for the various regions of the gel. We spent significant time testing different fabrication options and the protocol described here produces the best devices for the cassettes listed in the Table of Materials and Reagents. If researchers use an alternative cassette system, they may find it necessary to tweak the vol...

Next generation sequencing (NGS) has had a profound impact on the way genetics research is conducted. Where researchers once focused on sequencing the genome of an entire species, it is now possible to sequence the genome of a single tumor or even a single cell in one experiment.1 NGS has also made it cost effective to sequence the RNA transcripts found within a cell, a collection of data known as the transcriptome. The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years.2,3,4 Unfortunately, standard protocols are incompatible and researchers must choose whether to sequence DNA or RNA for a given sample. When a starting sample is large enough, it can be split in half. At smaller scales, however, loss of material due to splitting samples can affect library quality, and pooling of samples can average out interesting variations between cells.5 Furthermore, researchers are increasingly interested in examining samples that cannot be split, such as single cells or small heterogeneous tumor biopsies.6
To address this problem, three protocols have recently been developed to sequence both DNA and RNA from the same starting sample: Gel-seq7, G&#38;T-seq8, and DR-seq9. This article presents a detailed protocol for Gel-seq, which can be used to simultaneously generate DNA and RNA libraries from as few as 100 cells at negligible added cost. The novel aspect of Gel-seq is the ability to separate DNA and RNA based exclusively on size using low cost hydrogel matrices. The core innovation of the Gel-Seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a combination of polyacrylamide membranes that take advantage of the size differences between these molecules. To put these size differences in context, consider how DNA and RNA are imaged: while DNA exists on the micron-scale and can be viewed using traditional microscopes, RNA exists on the nanometer scale and must be imaged using complex techniques such as cryo-electron microscopy.10
The approach to separating DNA and RNA in this protocol is shown in Figure 1. The left panel shows DNA and RNA free floating in solution near a membrane. When an electric field is applied, as shown in the right panel, DNA and RNA experience an electrophoretic force that induces migration through the membrane. By tuning the membrane properties, we have created a semi-permeable membrane that separates DNA from RNA. The DNA molecules are pushed against the membrane, but become entangled at the edge because of their large size. Small RNA molecules, on the other hand, can reconfigure and weave their way through the membrane. This process, known as reptation, is similar to the way a snake moves through grass. Eventually these RNA molecules are stopped by a second, high-density membrane that is too difficult for even smaller polymers (&#62;200 base pairs) to wriggle through. Once physically separated, DNA and RNA can be recovered and processed to generate information about both the genome and transcriptome. While we can separate DNA and RNA, we have found better results are obtained if the RNA is reverse transcribed to cDNA before separation. The cDNA/RNA hybrids are more stable than RNA alone and can still pass through the low-density membrane.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57315/57315fig1.jpg" /> 
Figure 1. Gel-seq Operating Principle.&#160;The underlying principle used to physically separate DNA and RNA. In an applied electric field, small RNA molecules migrate through the low-density membrane but large DNA molecules are trapped at the surface. This figure was reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="//cloudflare.jove.com/files/ftp_upload/57315/57315fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This paper describes in detail both the fabrication of the Gel-seq device and the biological protocol to generate paired DNA and RNA libraries. An overview of both is shown in Figure 2. The device is fabricated by layering three different density polyacrylamide gels on top of each other in a process similar to creating standard stacking gels.11 The biological protocol starts with 100 - 1000 cells suspended in PBS. The cells are lysed and the RNA is converted into cDNA before the device is used to separate the genomic DNA from the cDNA/RNA hybrids. After separation and recovery, genomic and transcriptomic libraries are prepared using a process that closely follows the standard whole-genome library preparation kit protocol. Further detail about the development and validation of Gel-seq can be read in the Lab on a Chip publication &#34;Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.&#34;7
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57315/57315fig2.jpg" /> 
Figure 2. Gel-seq Protocol.&#160;An overview of the steps to fabricate the Gel-seq device and the protocol to generated paired DNA and RNA libraries. Portions of this figure were reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="//cloudflare.jove.com/files/ftp_upload/57315/57315fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To generate DNA and RNA libraries from single cells, researchers should consider using either G&#38;T-seq or DR-seq. G&#38;T-Seq, like Gel-seq, relies on a physical separation of RNA from genomic DNA. This approach relies on messenger RNA&#39;s (mRNA) 3&#8242; polyadenylated tail as a pull-down target. The mRNA is captured on a magnetic bead using a biotinylated oligo-dT primer. Once the mRNA has been captured the beads are held in place with a magnet and the supernatant containing the genomic DNA can be removed and transferred to another tube. After this physical separation is complete, separate libraries can be generated from the mRNA and DNA.8 This approach works well if the RNA of interest is polyadenylated, however it cannot be used to study non-polyadenylated transcripts, such as ribosomal RNA, tRNA, or RNA from prokaryotes.
DR-seq relies on a pre-amplification step where both DNA and cDNA derived from RNA are amplified in the same tube. The sample is then split in two and processed in parallel to prepare DNA- and RNA-seq libraries. To distinguish between genomic DNA and the cDNA derived from RNA, DR-seq takes a computational approach. Sequences where only exons are present are computationally suppressed in the genomic DNA data, as those could have originated from either DNA or RNA.9 An advantage of this approach is that the DNA and cDNA/RNA need not be physically separated as is done in Gel-seq and G&#38;T-seq. The drawback, however, is that DR-seq requires a priori knowledge of the genome and transcriptome (i.e., exons versus introns), and might not be ideal for applications such as sequencing of nuclei, in which many transcripts are not yet fully spliced and still contain introns.12
The novel aspect of Gel-seq is the ability to separate DNA and RNA in hundreds of cells based exclusively on size. This method requires no a priori knowledge of the genome or transcriptome, is robust against incomplete splicing, and is not limited to poly-adenylated transcripts. For applications where a researcher can start with at least 100 cells, Gel-seq provides a straightforward approach using cheap and widely-available materials.

<table border="0" cellpadding="0" cellspacing="0" style="width:905px;" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Reagents</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acrylamide Monomer</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A8887-100G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ammonium Persulfate</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A3678-25G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Ampure XP Beads</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:369px;">Referred to in the text as solid phase reversible immobilization (SPRI) beads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA Gel Loading Dye (6x)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td style="width:369px;">Referred to in the text as 6X loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ethyl alcohol</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">E7023-500ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">KAPA SYBR FAST One-Step qRT-PCR Kits</td>
			<td style="width:147px;">Kapa BioSystems</td>
			<td style="width:119px;">7959613001</td>
			<td style="width:369px;">Referred to in the text as 2X qPCR mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">N,N&#8242;-Methylenebis(acrylamide)&#160;</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">146072-100G</td>
			<td style="width:369px;">Also known as bis-acrylamide</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:271px;">NexteraXT DNA Library Preparation Kit (referred to in the text as library preparation kit)</td>
			<td style="width:147px;">Illumina</td>
			<td style="width:119px;">FC-131-1024</td>
			<td style="width:369px;">Includes: TD (referred to in the text as transposase buffer), ATM (referred to in the text as transposase), NT (referred to in the text as transposase stop buffer), and NPM (Referred to in the text as library prep PCR mix)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Nuclease Free Water</td>
			<td style="width:147px;">Millipore</td>
			<td style="width:119px;">3098</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Protease</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">19155</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:271px;">SMART-Seq v4 Kit (referred to in the text as whole-transcript amplification (WTA) kit)</td>
			<td style="width:147px;">Takara/Clontech</td>
			<td style="width:119px;">634888</td>
			<td style="width:369px;">Includes: Lysis buffer, RNase inhibitor, 3&#8217; SMART-Seq CDS Primer II A (referred to in the text as RT primer), 5X Ultra Low First Strand Buffer (referred to in the text as first strand buffer), SMART-Seq v4 Oligonucleotide (referred to in the text as template switch oligonucleotide (TSO)), SMART-Scribe Reverse Transcriptase (referred to in the text as reverse transcriptase), and PCR Primer II A (referred to in the text as cDNA PCR primer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Random hexamer with WTA adapter&#160;</td>
			<td style="width:147px;">IDT</td>
			<td style="width:119px;">n/a</td>
			<td>5&#8242;-AAGCAGTGGTATCAACGCAGAGTAC-NNNNNN-3&#8242;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sucrose</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">S0389-500G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">TEMED</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T9281-25ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA AWAY Surface Decontaminant</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">7010PK&#160;&#160;</td>
			<td style="width:369px;">Referred to in the text as DNA removal product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tris-Borate-EDTA buffer (10X concentration)</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T4415-1L</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">SYBR Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11494</td>
			<td style="width:369px;">Referred to in the text as gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;"></td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">BD Precisionglide&#160;syringe needles, gauge 18</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z192554</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Branson CPX series ultrasonic bath</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z769363</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Empty Gel Cassettes, mini, 1.0 mm</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NC2010</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Mesh Filter Plate - Corning&#160;HTS Transwell 96 well permeable supports - 8.0 &#181;m pore size</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">CLS3374</td>
			<td style="width:369px;">Referred to in the text as 8 um mesh filter plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PowerPac HC Power Supply</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1645052</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Qubit Fluorometer</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">Q33216</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vacufuge Concentrator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">22822993</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">XCell SureLock Mini-Cell system</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EI0001</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Bio-Rad CFX96 Touch Real-Time PCR Detection System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1855195&#160;</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Amersham UVC 500 Ultraviolet Crosslinker</td>
			<td style="width:147px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">UVC500-115V</td>
			<td style="width:369px;">Discontinued, any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Gel Doc XR+ Gel Documentation System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1708195</td>
			<td style="width:369px;">Referred to in the text as gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Dark Reader Transilluminator</td>
			<td style="width:147px;">Clare Chemical Research</td>
			<td style="width:119px;">DR89</td>
			<td style="width:369px;">Referred to in the text as UV transilluminator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">&#160;Ultrasonic&#160;Bath</td>
			<td style="width:147px;">Bransonic</td>
			<td style="width:119px;">1207K35</td>
			<td style="width:369px;">Any equivalent ultrasonic bath is acceptable.</td>
		</tr>
	</tbody>
</table>

gel-seq: a method for simultaneous sequencing library preparation of dna and rna using hydrogel matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

The physical separation of gDNA and cDNA/RNA hybrids in the Gel-seq device can be visualized through fluorescent gel imaging; a representative result is shown in Figure 3. Panel A shows the fabricated Gel-seq device; false color has been added to distinguish the different gel regions. Panel B shows a close up of four different separations used for validation. The third lane, a negative control, represents background and shows that there is no autoflourescence...

Watch this Scientific Journal Video about Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices at JoVE.com

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

The overall goal of this experiment is to generate next generation sequencing libraries from both the DNA and RNA of small populations of cells without splitting the sample at any point. This method provides researchers with a new approach to study the structural variations of DNA on the expression of genes. When we first thought up Gel-seq, there were no protocols for simultaneously sequencing both the DNA and RNA from the same sample.Fortunately, there are now three published approaches that solve this problem. The main advantage of this technique is that both genomic and transcriptomic information can be obtained from the same set of cells for negligible added cost. Gel-seq was originally developed with upright cassettes, however, this protocol can be adapted to work with any standard gel electrophoresis cassette.Prepare the filler, the high density, and the low density precursor mixtures in three tubes. Add the reagents in the order described, and vortex the mixture. Do not prematurely add either APS or Temed.Wait until directed. Next, degas each gel monomer solution by first inserting a needle through the cap. Then connecting the needle to a house vacuum line, and submerging this assembly in an ultra sonic bath.Wait about one minute until no more bubbles emerge from the liquid. Next, to the filler precursor mixture, add the Temed and the APS and vortex briefly. Within three minutes, use a one millimeter pipette to add six millimeters to each gel cassette without spilling.Ensure the fluid is level by positioning the cassettes upright on a level table. Then, gradually top off the cassettes with degassed, ionized water, being careful to minimize the mixing of the polymerizing mixture with the water. Then, allow the polymer to cure for an hour to overnight.Once the polymer is cured, dump out the water overlay, and from six inches away use a gentle stream of compressed air to dry the gel's surface. Next, add Temed and APS to the high density gel precursor, and vortex briefly. Within three minutes, transfer 320 microliters to each cassette, and spread it over the filler gel layer by rocking the cassette back and forth a few times.Then, top off the cassettes with deionized, degassed water, and allow the polymer to cure for about an hour. Once cured, dry off the polymer as before. Finally, add Temed and APS to the low density gel precursor mixture, and vortex it.Then, quickly fill the remainder of the cassettes with this mix, and insert the gel combs before it starts to cure. Now, add the excess of reserve precursor over the comb to be adsorbed during polymerization. After curing, the cassette is ready for use.After converting the RNA to cDNA, use a gel to recover the fraction of interest. First, thoroughly clean the electrophoresis chamber using several milliliters of a DNA removal product, and a lint-free wipe. Then, fill the chamber with clean, zero point five XTBE, and place the entire apparatus in a 254 nanometer, UV-crosslinking oven, and sterilize for 15 minutes.Now, insert a Gel-seq cassette into the electrophoresis chamber and lock it in place. Then, slowly remove the gel comb by gently pulling upwards without tearing the gel. Now, load samples using a pipette.Fully insert the tip into the wells when loading samples to minimize the chance of contamination between wells. Apply an electric field of 250 volts across the Gel-seq cassette for 30 minutes to separate the gDNA from the cDNA/RNA hybrids. After loading, running, and collecting the gel use a scalpel to cut the gel in half just below the high density layer, and discard the filler gel half.Then, gently peel the remaining gel off of the cassette making careful use of a scraping tool. After verifying separation using a gel doc, use a transilluminator to excise the bands. The gDNA should be located at the start of the low density gel, and the cDNA should be at the interface of the low and high density regions.Now, use a scalpel to cut out the regions of the gel containing gDNA and cDNA. Also remove the same sections from the negative control lane. Using blunted tweezers, gently transfer the sections of the gel into strip tubes.If the gel breaks, find and transfer all the broken pieces to the tube. Next, grind down each gel using circular movements, and pressing with a pipette tip. Before removing the tips, add 40 microliters of nuclease-free water to the gDNA, and 80 microliters to the cDNA samples.Then, carefully remove and discard the tips. Now, secure the strip tubes to a vortex mixer at 37 degrees Celsius and shake them for eight to 12 hours. The DNA will thus elude from the gel.After the shaking, pipette the samples into an eight micron mesh filter plate. Then, spin the plate at 2, 600 g for five minutes to strain out the gel fragments. Next, lift the mesh filter plate away from the housing plate and use a pipette to transfer the gel-free samples from the plate to new 200 microliter strip tubes.To each sample containing gDNA, add one microliter of protease, and pipette up and down to mix well. Then, incubate the samples at 50 degrees Celsius for 15 minutes. Follow with a heating activation at 70 degrees Celsius for 15 minutes.This nucleosome depletion step is critical. Next, use an 18 gauge needle to poke a hole in the cap of every sample tube. Then, use a vacufuge to reduce the gDNA samples to five microliters, and the cDNA samples to 10 microliters.If the volume drops below the target, add nuclease-free water as needed. gDNA and cDNA/RNA hybrids were physically separated using the Gel-seq device. The negative control has no auto fluorescence of the gel at the interfaces.The DNA ladders show only a dark band at the interface between the low and high density membranes, revealing that small fragments can pass through the low density gel. A biological sample from 500 PC3 cells shows the desired separation of genomic DNA and cDNA/RNA hybrids. A dark band at the top of the low density membrane is megabase-scale genomic DNA while the cDNA/RNA hybrids are stacked at the interface of the low and high density regions.The small sub-100 base pair fragments are off target products generated from primer oligonucleotides during reverse transcription and are to be expected. Libraries were generated from 500 PC3 cells or 750 HeLa cells. Fragment distributions from the matched libraries generated from Gel-seq were compared to unmatched samples generated with standard protocols.The distribution of nucleotide fragments was similar with either method. Sequencing results of the PC3 cell library were compared with traditional methods. Genome-wide copy number variations were compared.Statistically, the two methods garnered similar results. After watching this video, you should have a good understanding of how to generate paired next generation sequencing libraries for both DNA and RNA from the same starting sample. As with any low input sequencing experiment, it's imperative to remember to maintain a clean working environment.You should pay careful attention to sterilizing tubes and cleaning gloves. Even with these precautions, experiments occasionally fail due to contamination. An important note, the ubiquity and safety of polyacrylamide gels makes it easy to forget that the fabrication precursors are dangerous neurotoxins.You should always work with these reagents in a fume hood and immediately clean up any spills. We designed Gel-seq to be simple for researchers to implement in their own labs, and we hope this will facilitate the rapid adoption of this technology.

gel-seq-method-for-simultaneous-sequencing-library-preparation-dna

Research

JoVE Journal

Bioengineering

8.9K ビュー.  University of San Diego. The overall goal of this experiment is to generate next generation sequencing libraries from both the DNA and RNA of small populations of cells without splitting the sample at any point. This method provides researchers with a new approach to study the structural variations of DNA on the expression of genes. When we first thought up Gel-seq, there were no protocols for simultaneously sequencing both the DNA and RNA from the same sample.Fortunately, there are now three published approac...

ビデオ: Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired...

ribo-seq in bacteria: a sample collection and library preparation protocol for ngs sequencing

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35...

interactome-seq: a protocol for domainome library construction, validation and selection by phage display and next generation sequencing

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data...

3' end sequencing library preparation with a-seq2

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing...

3' End Sequencing Library Preparation with A-seq2

rare event detection using error-corrected dna and rna sequencing

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as...

preparation of ffpe dna and rna samples for sequencing using an automated purification instrument (api)

Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument (API)  

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality...

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

Author Spotlight: Decoding RNA Methylation&#039;s Role in Pancreatic Cancer - A Single-Base Resolution Study 

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of...

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

atac-seq library preparation of murine bone marrow-derived neutrophils

Author Spotlight: Optimizing ATAC-seq Library Construction for Investigating Neutrophil Chromatin Accessibility and Epigenetic Reprogramming in Rodent Models

This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying...

Scalable ATAC-seq Protocol for Chromatin Accessibility in Rodent Neutrophils

https://cloudfront.jove.com/CDNSource/teasers/67490_b.jpg

a bioinformatics pipeline for investigating molecular evolution and gene expression using rna-seq

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing...

circle-seq for interrogation of off-target gene editing

Author Spotlight: Developing iPSC-Based Therapies for Genetic Skin Diseases Using Precision Gene Editing

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

https://cloudfront.jove.com/CDNSource/teasers/67069_b.jpg

single-cell rna sequencing of fluorescently labeled mouse neurons using manual sorting and double in vitro transcription with absolute counts sequencing (diva-seq)

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

ultralow input genome sequencing library preparation from a single tardigrade specimen

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of...

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

2d-hels ms seq: a general lc-ms-based method for direct and de novo sequencing of rna mixtures with different nucleotide modifications

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base...

aqrna-seq for quantifying small rnas

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced...

Analyzing tRNA Dynamics Using AQRNA-seq

https://cloudfront.jove.com/CDNSource/teasers/66335_b.jpg

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

damid-seq: genome-wide mapping of protein-dna interactions by high throughput sequencing of adenine-methylated dna fragments

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq,...

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of...

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...