Name: gel-seq: a method for simultaneous sequencing library preparation of dna and rna using hydrogel matrices
Uploaded: 2018-03-26T14:00:00
Description: 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

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.

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