Name: transcription start site mapping using super-low input carrier-cage
Uploaded: 2019-06-26T15:00:00
Duration: 6 min 59 s
Description: Watch this Scientific Journal Video about Transcription Start Site Mapping Using Super-low Input Carrier-CAGE at JoVE.com

This work was supported by The Wellcome Trust grant (106954) awarded to B. L. and Medical Research Council (MRC) Core Funding (MC-A652-5QA10). N. C. was supported by EMBO Long-Term Fellowship (EMBO ALTF 1279-2016); E. P. was supported by the Medical Research Council UK; B. L. was supported by the Medical Research Council UK (MC UP 1102/1).

<ol>
	<li>Shiraki, T., 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=Cap+analysis+gene+expression+for+high-throughput+analysis+of+transcriptional+starting+point+and+identification+of+promoter+usage.">Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (26), 15776-15781 (2003).</li><li>Haberle, V., Lenhard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+architectures+and+developmental+gene+regulation.">Promoter architectures and developmental gene regulation.</a> Seminars in Cell and Developmental Biology. 57, 11-23 (2016).</li><li>Haberle, V., Stark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+core+promoters+and+the+functional+basis+of+transcription+initiation.">Eukaryotic core promoters and the functional basis of transcription initiation.</a> Nature Reviews Molecular Cell Biology. 19 (10), 621-637 (2018).</li><li>Andersson, R., 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=An+atlas+of+active+enhancers+across+human+cell+types+and+tissues.">An atlas of active enhancers across human cell types and tissues.</a> Nature. 507 (7493), 455-461 (2014).</li><li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Celniker, S. E., 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=Unlocking+the+secrets+of+the+genome.">Unlocking the secrets of the genome.</a> Nature. 459 (7249), 927-930 (2009).</li><li>Consortium, F., 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=A+promoter-level+mammalian+expression+atlas.">A promoter-level mammalian expression atlas.</a> Nature. 507 (7493), 462-470 (2014).</li><li>Boyd, M., 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=Characterization+of+the+enhancer+and+promoter+landscape+of+inflammatory+bowel+disease+from+human+colon+biopsies.">Characterization of the enhancer and promoter landscape of inflammatory bowel disease from human colon biopsies.</a> Nature Communications. 9 (1), 1661 (2018).</li><li>Adiconis, X., 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=Comprehensive+comparative+analysis+of+5'-end+RNA-sequencing+methods.">Comprehensive comparative analysis of 5'-end RNA-sequencing methods.</a> Nature Methods. , (2018).</li><li>Cvetesic, 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=SLIC-CAGE:+high-resolution+transcription+start+site+mapping+using+nanogram-levels+of+total+RNA.">SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.</a> Genome Research. 28 (12), 1943-1956 (2018).</li><li>Murata, M., 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=Detecting+expressed+genes+using+CAGE.">Detecting expressed genes using CAGE.</a> Methods in Molecular Biology. 1164, 67-85 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9, 357 (2012).</li><li>A language and environment for statistical computing. Available from: <a target="_blank" href="https://www.R-project.org/">https://www.R-project.org/</a> (2017)</li><li>Haberle, V., Forrest, A. R., Hayashizaki, Y., Carninci, P., Lenhard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAGEr:+precise+TSS+data+retrieval+and+high-resolution+promoterome+mining+for+integrative+analyses.">CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses.</a> Nucleic Acids Research. 43 (8), e51 (2015).</li><li>Poulain, S., 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=NanoCAGE:+A+Method+for+the+Analysis+of+Coding+and+Noncoding+5'-Capped+Transcriptomes.">NanoCAGE: A Method for the Analysis of Coding and Noncoding 5'-Capped Transcriptomes.</a> Methods in Molecular Biology. 1543, 57-109 (2017).</li><li>Schon, M. A., Kellner, M. J., Plotnikova, A., Hofmann, F., Nodine, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoPARE:+parallel+analysis+of+RNA+5'+ends+from+low-input+RNA.">NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.</a> Genome Research. 28 (12), 1931-1942 (2018).</li></ol>

A patent for degradable carrier RNA/DNA has been filled.

For successful SLIC-CAGE library preparations, it is critical to use low-binding tips and tubes to prevent sample loss due to sample adsorption. In all steps involving retrieval of the supernatant, it is recommended to recover the entiresample volume. As the protocol has multiple steps, continuous sample loss will lead to unsuccessful libraries.
If CAGE (nAnT-iCAGE) has not been performed routinely, it is best to test SLIC-CAGE with different input amounts (10 ng, 20 ng, 50 ng, 100 ng, 200 ng) of the same total RNA sample and compare to nAnT-iCAGE libraries that are prepared using 5 &#181;g of total RNA. If the nAnT-iCAGE library is unsuccessful (less than 0.5-1 ng of the DNA library obtained per sample), SLIC-CAGE is unlikely to work, and sample loss needs to be minimized.
A critical step to ensure high quality libraries devoid of uncapped degraded RNA or rRNA is the cap-trapping described in section 7. It is highly important that the streptavidin beads are thoroughly resuspended in wash buffers and that the wash buffers are removed prior to continuing to the next wash step or elution of cDNA.
If results from the qPCR after the first round of carrier degradation show no difference between the use of adaptor_f1 and carrier_f1 primers, continuing the protocol is still recommended. If after the second round of carrier degradation, the difference in Ct values is less than five, a third round of carrier degradation is recommended. We have never found a third round of degradation necessary, and if it occurs, it is recommended to replace the homing endonuclease stocks.
Additional rounds of PCR amplification may be added to the protocol if the final amount of the library obtained is not enough for sequencing. PCR amplification can then be set with minimal number of amplification cycles needed to yield enough material for sequencing, taking into account sample loss that cannot be avoided in size selection. Purification or size selection using SPRI magnetic beads should then be performed until all small (&#60;200 bp) fragments are removed (if needed, use 0.6:1 beads to sample ratio), and the library should be quantified using Picogreen.
Libraries can be sequenced in single-end or paired-end mode. Using paired-end sequencing, information about transcript isoforms can be obtained. In addition, as reverse transcription is performed using a random primer (TCT-N6, N6 being a random hexamer), information from the sequenced 3&#8217;-end can be used as unique molecular identifiers (UMI) to collapse PCR duplicates. As a moderate number of PCR amplification cycles is used (up to 18), the use of UMIs&#160;has been previously found to be unnecessary.
As the core of the protocol relies on nAnT-iCAGE11, SLIC-CAGE uses eight barcodes. Therefore, multiplexing more than eight samples is currently not supported. In addition, both SLIC-CAGE and nAnT-iCAGE are not suitable for capturing RNAs shorter than 200 bp, as the protocols are designed to remove linkers and PCR artefacts through size-exclusion with AMPure XP beads.
SLIC-CAGE is the only unbiased low-input single-nucleotide resolution method for mapping transcription initiation start sites using nanograms of total RNA material. Alternative methods rely on the template switching activity of the reverse transcriptase to barcode capped RNA instead of cap-trapping (e.g., NanoCAGE15 and NanoPARE16). Due to template switching, these methods exhibit sequence-specific biases in TSSs detection, leading to increased numbers of false positive TSSs and decreased numbers of true TSSs9,10.

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution genome-wide mapping of RNA polymerase II transcription start sites (TSSs)1. Its quantitative nature also allows 5&#8217;-end centric expression profiling. Regions surrounding the TSSs (about 40 bp upstream and downstream) are core promoters and represent the physical location where RNA polymerase II and general transcription factors bind (reviewed previously2,3). Information on exact locations of TSSs can be used for core promoter discovery and for monitoring promoter dynamics. In addition, as active enhancers exhibit signatures of bidirectional transcription, CAGE data can also be used for enhancer discovery and monitoring of enhancer dynamics4. CAGE methodology has recently increased in popularity due to its broad application and use in high-profile research projects such as ENCODE5, modENCODE6, and FANTOM projects7. In addition, TSS information is also proving to be important for distinguishing healthy and diseased tissue, as disease-specific TSSs can be used for diagnostic purposes8.
Even though several methods for TSS mapping are available (CAGE, RAMPAGE, STRT, nanoCAGE, nanoCAGE-XL, oligo-capping), we and others have recently shown that CAGE is the most unbiased method to capture true TSSs with the least number of false positives9,10. The recent CAGE protocol, nAnT-iCAGE11, is the most unbiased protocol for TSS profiling, as it avoids cutting the fragments to short tags using restriction enzymes and does not use PCR amplification. A limitation of the nAnT-iCAGE protocol is the requirement for a large amount of starting material (e.g., 5 &#181;g&#160;of total RNA for each sample). To answer specific, biologically relevant questions, it is often impossible to obtain such high amounts of starting material (e.g., for FACS-sorted cells or early embryonic stages). Finally, if nAnT-iCAGE is successful, only 1-2 ng of DNA library material is available from each sample, thereby limiting the achievable sequencing depth.
To enable TSS profiling using only nanograms of total RNA, we have recently developed Super-low Input Carrier-CAGE10 (SLIC-CAGE, Figure 1). SLIC-CAGE requires only 10 ng of total RNA to obtain high complexity libraries. Our protocol relies on the carefully designed synthetic RNA carrier added to the RNA of interest to achieve a total of 5 &#956;g of RNA material. The synthetic carrier mimics the target DNA library in length distribution to avoid potential biases that could be caused by homogenous molecules in excess. The sequence of the carrier is based on the sequence of the Escherichia coli leucyl-tRNA synthetase gene (Table 1) for two reasons. First, any leftover of the carrier in the final library, even if sequenced, will not map to a eukaryotic genome. Second, as E. coli is a mesophilic species, its housekeeping genes are optimised for the temperature range appropriate for SLIC-CAGE. The carrier sequence is also embedded with homing endonuclease recognition sites to allow specific degradation of DNA derived from the carrier RNA molecules. The target, sample-derived library remains intact, as the homing endonuclease recognition sites are long (I-CeuI = 27 bp; I-SceI = 18 bp) and statistically unlikely to be found in eukaryotic genomes. After specific degradation of the carrier and removal of fragments by size exclusion, the target library is PCR amplified and ready for next-generation sequencing. Depending on the starting RNA amount (1-100 ng), between 13-18 PCR amplification cycles are expected to be required. The final amount of DNA per each sample ranges between 5-50 ng, yielding enough material for very deep sequencing. When using only 1-2 ng of total RNA, true TSSs can be detected; however, the libraries are expected to be of lower complexity. Lastly, as SLIC-CAGE is based on the nAnT-iCAGE protocol11, it enables multiplexing of up to eight samples prior to sequencing.

<table>
	<tbody>
		<tr>
			<td>2-propanol, Bioultra, for molecular biology, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>59304-100ML-F</td>
			<td>Used in RNAclean XP purification.</td>
		</tr>
		<tr>
			<td>3&#39; linkers</td>
			<td></td>
			<td></td>
			<td>Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 3&#39;linkers is described in the supplementary of this protocol.</td>
		</tr>
		<tr>
			<td>5&#39; linkers</td>
			<td></td>
			<td></td>
			<td>Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 5&#39;linkers is described in the supplementary of this protocol.</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP, 60 mL</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Purification of DNA</td>
		</tr>
		<tr>
			<td>Agencourt RNAClean XP Kit</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>Purification of RNA and RNA:cDNA hybrids in CAGE steps.</td>
		</tr>
		<tr>
			<td>Axygen 0.2 mL Polypropylene PCR Tube Strips and Domed Cap Strips</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-0208-CP-C</td>
			<td>Or any 8-tube PCR strips (used only for water and mixes).</td>
		</tr>
		<tr>
			<td>Axygen 1 x 8 strip domed PCR caps</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-02CP-C</td>
			<td>Caps for PCR plates.</td>
		</tr>
		<tr>
			<td>Axygen 1.5 mL Maxymum Recovery Snaplock Microcentrifuge Tube</td>
			<td>Axygen (available through Corning)</td>
			<td>MCT-150-L-C</td>
			<td>Low-binding 1.5 ml tubes, used for enzyme mixes or sample concentration.</td>
		</tr>
		<tr>
			<td>Axygen 96 well no skirt PCR microplate</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-96-C</td>
			<td>Low-binding PCR plates - have to be used for all steps in the protocol. Note that plates should be cut to contain 2 x 8 wells for easier visibility of the samples</td>
		</tr>
		<tr>
			<td>Bioanalyzer (or Tapestation): RNA nano and HS DNA kits</td>
			<td>Agilent</td>
			<td></td>
			<td>To determine quality of RNA, efficient size selection and final quality of the library (Tapestation can also be used)</td>
		</tr>
		<tr>
			<td>Biotin (Long Arm) Hydrazide</td>
			<td>Vector laboratories</td>
			<td>SP-1100</td>
			<td>Biotinylation/tagging</td>
		</tr>
		<tr>
			<td>Cutsmart buffer</td>
			<td>NEB</td>
			<td></td>
			<td>Restriction enzyme buffer</td>
		</tr>
		<tr>
			<td>Deep Vent (exo-) DNA Polymerase</td>
			<td>NEB</td>
			<td>M0259S</td>
			<td>Second strand synthesis</td>
		</tr>
		<tr>
			<td>DNA Ligation Kit, Mighty Mix</td>
			<td>Takara</td>
			<td>6023</td>
			<td>Used for 5&#39; and 3&#39;-linker ligation</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM each)</td>
			<td>ThermoFisher Scientific</td>
			<td>18427013</td>
			<td>dNTP mix for production of carrier templates (or any dNTPs suitable for PCR)</td>
		</tr>
		<tr>
			<td>Dynabeads M-270 Streptavidin</td>
			<td>Invitrogen</td>
			<td>65305</td>
			<td>Cap-trapping. Do not use other beads as these are optimised with the buffers used.</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12321D</td>
			<td>Magnetic stand for 1.5 ml tubes - used to prepare Streptavidin beads.</td>
		</tr>
		<tr>
			<td>DynaMag-96 Side Skirted Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12027</td>
			<td>Magnetic stand for PCR plates (96 well-plates) - used with cut plates to contain 2 x 8 wells.</td>
		</tr>
		<tr>
			<td>Ethanol, BioUltra, for molecular biology, &#8805;99.8%</td>
			<td>Sigma-Aldrich</td>
			<td>51976-500ML-F</td>
			<td>Used in AMPure washes. Any molecular biology suitable ethanol can be used.</td>
		</tr>
		<tr>
			<td>Exonuclease I (E. coli)</td>
			<td>NEB</td>
			<td>M0293S</td>
			<td>Leftover primer degradation</td>
		</tr>
		<tr>
			<td>Gel Loading Dye, Purple (6x), no SDS</td>
			<td>NEB</td>
			<td>B7025S</td>
			<td>agarose gel loading dye</td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2040S</td>
			<td>Kit for carrier in vitro transcription</td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis apparatus</td>
			<td></td>
			<td></td>
			<td>purification of carrier DNA templates from agarose gels</td>
		</tr>
		<tr>
			<td>I-Ceu</td>
			<td>NEB</td>
			<td>R0699S</td>
			<td>Homing endonuclease used for carrier degradation.</td>
		</tr>
		<tr>
			<td>I-SceI</td>
			<td>NEB</td>
			<td>R0694S</td>
			<td>Homing endonuclease used for carrier degradation.</td>
		</tr>
		<tr>
			<td>KAPA HiFi HS ReadyMix (2x)</td>
			<td>Kapa Biosystems (Supplied by Roche)</td>
			<td>KK2601</td>
			<td>PCR mix for target library amplification</td>
		</tr>
		<tr>
			<td>KAPA SYBR FAST qPCR kit (Universal) 2x</td>
			<td>Kapa Biosystems (Supplied by Roche)</td>
			<td>KK4600</td>
			<td>qPCR mix to assess degradation efficiency and requiered number of PCR amplification cycles</td>
		</tr>
		<tr>
			<td>Micropipettes and multichannel micropipettes (0.1-10 &#181;l, 1-20 &#181;l, 20-200 &#181;)</td>
			<td>Gilson</td>
			<td></td>
			<td>Use of Gilson with the low-binding Sorenson tips is recommended. Other micropippetes might not be compatible.. Different brand low-binding tips may not be of equal quality and may increase sample loss.</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td></td>
			<td></td>
			<td>For Picogreen concentration measurement of the final library. Microplates are used to allow small volume measurement and reduce sample waste.</td>
		</tr>
		<tr>
			<td>nuclease free water</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9937</td>
			<td>Or any nuclease (DNase and RNase) free water</td>
		</tr>
		<tr>
			<td>PCR thermal cycler</td>
			<td></td>
			<td></td>
			<td>incubation steps and PCR amplficication</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>F530S</td>
			<td>DNA polymerase for amplification of carrier templates (or any high fidelity polymerase)</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Purification of carrier PCR templates from agarose gels.</td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td></td>
			<td></td>
			<td>determining PCR amplification cyle number and degree of carrier degradation</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>P11495</td>
			<td>Used to measure final library concentration - recommended as, in our hands, it is more accurate and reproducible than Qubit.</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 100 bp DNA Ladder</td>
			<td>NEB</td>
			<td>N0551S</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 1 kb Plus DNA Ladder</td>
			<td>NEB</td>
			<td>N0550S</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>Ribonuclease H</td>
			<td>Takara</td>
			<td>2150A</td>
			<td>Digestion of RNA after cap-trapping.</td>
		</tr>
		<tr>
			<td>RNase ONE Ribonuclease</td>
			<td>Promega</td>
			<td>M4261</td>
			<td>Degradation of single stranded RNA not protected by cDNA.</td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>Removal of carrier DNA templates after in vitro transcription.</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>For cleanup of carrier RNA from in vitro transcription or capping</td>
		</tr>
		<tr>
			<td>Sodium acetate, 1 M, aq.soln, pH 4.5 RNAse free</td>
			<td>VWR</td>
			<td>AAJ63669-AK</td>
			<td>Or any nuclease (DNase and RNase) free solution</td>
		</tr>
		<tr>
			<td>Sodium acetate, 1 M, aq.soln, pH 6.0 RNAse free</td>
			<td></td>
			<td></td>
			<td>Or any nuclease (DNase and RNase) free solution</td>
		</tr>
		<tr>
			<td>Sodium periodate</td>
			<td>Sigma-Aldrich</td>
			<td>311448-100G</td>
			<td>Oxidation of vicinal diols</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MicroReach Guard, volume range 10 &#956;L, Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719390-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 1000 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719463-1000EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 20 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719412-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 200 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719447-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>SpeedVac Vacuum Concentrator</td>
			<td></td>
			<td></td>
			<td>concentrating samples in various steps to lower volume</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080044</td>
			<td>Used for reverse transcription (1st CAGE step)</td>
		</tr>
		<tr>
			<td>Trehalose/sorbitol solution</td>
			<td></td>
			<td></td>
			<td>Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Tris-HCl, 1M aq.soln, pH 8.5</td>
			<td></td>
			<td></td>
			<td>1 M solution, DNase and RNase free</td>
		</tr>
		<tr>
			<td>tRNA (20 mg/mL)</td>
			<td></td>
			<td></td>
			<td>tRNA solution. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16520050</td>
			<td>Or any suitable pure low-melt agarose.</td>
		</tr>
		<tr>
			<td>USB Shrimp Alkaline Phosphatase (SAP)</td>
			<td>Applied Biosystems (Provided by ThermoFisher Scientific)</td>
			<td>78390500UN</td>
			<td></td>
		</tr>
		<tr>
			<td>USER Enzyme</td>
			<td>NEB</td>
			<td>M5505S</td>
			<td>Degradation of 3&#39;linker&#39;s upper strand, Uracil Specific Excision Reagent/Enzyme</td>
		</tr>
		<tr>
			<td>Vaccinia Capping System</td>
			<td>NEB</td>
			<td>M2080S</td>
			<td>Enzymatic kit for in vitro capping of carrier molecules</td>
		</tr>
		<tr>
			<td>Wash buffer A</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Wash buffer B</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Wash buffer C</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
	</tbody>
</table>

transcription start site mapping using super-low input carrier-cage

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 &#176;C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5&#8217;-centric expression profiling.

This report describes the full SLIC-CAGE protocol for obtaining sequencing-ready libraries from nanograms of starting total RNA material (Figure 1). To obtain the synthetic RNA carrier mix, first, PCR carrier templates need to be prepared and gel-purified to eliminate PCR side products (Figure 2A). Each PCR template (ten in total) is produced by using a common forward, but a different reverse primer (Table 2), leading to different lengths of the PCR template to enable size variability of synthetic RNA carriers. Once purified, PCR templates are used for in vitro transcription of the carrier molecules. A single RNA carrier product is expected if the templates are gel-purified (see representative gel-analysis in Figure 2B). Preparation of the carrier can be upscaled depending on the need, and when prepared, mixed and frozen at -80 &#176;C for future use.
Using the recommended minimal amount of sample total RNA (10 ng) combined with 16-18 cycles of PCR amplification, high complexity SLIC-CAGE libraries can be achieved. Number of PCR cycles required to amplify the final library highly depends on the amount of total input RNA used (the expected number of cycles is presented in Table 4).
After the first round of degradation, in qPCR results (step 17), the expected difference between Ct values obtained using adaptor_f1 or carrier_f1 primer is 1-2, with Ct values obtained with adaptor_f1 lower than with carrier_f1.
The distribution of the fragment lengths in the final library is between 200-2,000 bp with the average fragment size of 700-900 bp (based on the region analysis using Bioanalyzer software, Figure 4B,D). Shorter fragments, as presented in Figure 4A,C, have to be removed by additional rounds of size-exclusion (steps 20-21). These short fragments are PCR amplification artefacts and not the target library. Note that shorter fragments cluster better on the sequencing flow cells and may cause sequencing problems.
The expected amount of library material obtained per sample is between 5-50 ng. Significantly lower amounts are indicative of sample loss during the protocol. If the obtained low quantity is enough for sequencing (2-3 ng of the pooled libraries is needed), the libraries may be of lower complexity (see below).
Depending on the sequencing machine, quantity of the library loaded onto the flow cell may need to be optimised. Using an Illumina HiSeq 2500, loading 8-12 pM SLIC-CAGE libraries gives on average 150-200 million reads, with &#62;80% of reads passing quality score Q30 as threshold.
The obtained reads are then mapped to the reference genome [for 50 bp reads, Bowtie212 can be used with default parameters that allow zero mismatches per seed sequence (22 bp)]. Expected mapping efficiencies depend on the total RNA input amount and are presented in Table 5. The uniquely mapped reads can then be loaded into R graphical and statistical computing environment13 and processed using CAGEr (Bioconductor package14). The package vignette is easy to follow and explains the workflow and processing of the mapped data in detail. An easy visual control of the library complexity is the distribution of promoter width, as low-complexity libraries will have artificially narrow promoters (Figure 5A, SLIC-CAGE library derived from 1 ng of total RNA, for details see previous publication10). However, even the low-complexity SLIC-CAGE libraries allow identification of true CTSSs, with greater precision than alternative methods for low/medium-input TSS mapping (Figure 5B,C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59805/59805fig1.jpg" /> 
Figure 1: Steps in the SLIC-CAGE protocol. Sample RNA is mixed with the RNA carrier mix to achieve 5 &#181;g of total RNA material. cDNA is synthesised through reverse transcription and the cap is oxidized using sodium periodate. Oxidation allows attachment of biotin to the cap using biotin hydrazide. Biotin gets attached to the mRNA&#8217;s 3&#8242; end, as it is also oxidized using sodium periodate. To eliminate biotin from mRNA:cDNA hybrids with incompletely synthesized cDNA and from the 3&#8242; ends of mRNA, the samples are treated with RNase I. cDNA that reached the 5&#8242; end of mRNA is then selected by affinity purification on streptavidin magnetic beads (cap-trapping). After release of cDNA, 5&#8242;- and 3&#8242;-linkers are ligated. The library molecules that originate from the carrier are degraded using I-SceI and I-CeuI homing endonucleases and the fragments are removed using SPRI magnetic beads. The library is then PCR amplified. <a href="https://www.jove.com/files/ftp_upload/59805/59805fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59805/59805fig2.jpg" /> 
Figure 2: Representative gel-analysis of carrier PCR templates and carrier in vitro transcripts. (A) Carrier PCR templates prior to gel purification: the first well contains the 1 kbp marker, followed by carrier PCR templates 1, 1-10. (B) Carrier in vitro transcripts: the first well contains the 1 kbp marker, followed by carrier transcripts 1-10. Carrier transcripts were denatured by heating for 5 min at 95 &#176;C prior to loading. <a href="https://www.jove.com/files/ftp_upload/59805/59805fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59805/59805fig3.jpg" /> 
Figure 3: Representative DNA quality (high sensitivity DNA chip) trace of SLIC-CAGE prior to first round of carrier degradation. <a href="https://www.jove.com/files/ftp_upload/59805/59805fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59805/59805fig4.jpg" /> 
Figure 4: Representative DNA quality (high sensitivity DNA chip) traces of SLIC-CAGE libraries after PCR amplification. (A) SLIC-CAGE library that requires additional size-selection for removal of short fragments. (B) SLIC-CAGE library after size-selection using 0.6x SPRI beads to sample ratio. (C) SLIC-CAGE library of lower output amount that requires size-selection for removal of short fragment. (D) SLIC-CAGE library of lower output amount after size-selection using 0.6:1 SPRI beads to sample ratio. <a href="https://www.jove.com/files/ftp_upload/59805/59805fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59805/59805fig5.jpg" /> 
Figure 5: Validation of SLIC-CAGE libraries. (A) Distribution of tag cluster interquantile widths in SLIC-CAGE libraries prepared from 1, 5, or 10 ng of S. cerevisiae total RNA, and in the nAnT-iCAGE library prepared from 5 &#181;g of S. cerevisiae total RNA. A high amount of narrow tag clusters in the 1 ng SLIC-CAGE library indicates its low complexity. (B) ROC curves for CTSS identification in S. cerevisiae SLIC-CAGE libraries. All S. cerevisiae nAnT-iCAGE CTSSs were used as a true set. (C) ROC curves for CTSS identification in S. cerevisiae nanoCAGE libraries. All S. cerevisiae nAnT-iCAGE CTSSs were used as a true set. Comparison of ROC curves shows that SLIC-CAGE strongly outperforms nanoCAGE in CTSS identification. Data from ArrayExpress E-MTAB-6519 was used. <a href="https://www.jove.com/files/ftp_upload/59805/59805fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequence of the carrier synthetic gene. I-SceI sites are bold and italicized in purple, and I-CeuI recognitions sites are green. <a href="https://www.jove.com/files/ftp_upload/59805/Table1.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>carrier </td>
			<td colspan="2">reverse primer 5&#8217;-3&#8217;</td>
			<td>PCR product length / bp</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="2">PCR_N6_r1: NNNNNNCTACGTGTCGCAGACGAATT</td>
			<td>1034</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="2">PCR_N6_r2: NNNNNNTATCCAGATCGTTGAGCTGC</td>
			<td>966</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="2">PCR_N6_r3: NNNNNNCACTGCGGGATCTCTTTACG</td>
			<td>889</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="2">PCR_N6_r4: NNNNNNGCCGTCGATAACTTGTTCGT</td>
			<td>821</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">PCR_N6_r5: NNNNNNAGTTGACCGCAGAAGTCTTC</td>
			<td>744</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="2">PCR_N6_r6: NNNNNNGTGAAGAATTTCTGTTCCCA</td>
			<td>676</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="2">PCR_N6_r7: NNNNNNCTCGCGGCTCCAGTCATAAC</td>
			<td>599</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="2">PCR_N6_r8: NNNNNNTATACGCGATGTTGTCGTAC</td>
			<td>531</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="2">PCR_N6_r9: NNNNNNACCGCCGCGCCTTCCGCAGG</td>
			<td>454</td>
		</tr>
		<tr>
			<td>10</td>
			<td colspan="2">PCR_N6_r10: NNNNNNCAGGACGTTTTTGCCCAGCA</td>
			<td>386</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">* Forward primer is the same for all carrier templates. Underlined is the T7 promoter sequence. PCR_GN5_f1: TAATACGACTCACTATAGNNNNNCAGCGTTCGCTA</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Primers for carrier template amplification. Forward primer is the same for all carrier templates. Underlined is the T7 promoter sequence. PCR_GN5_f1: TAATACGACTCACTATAGNNNNNCAGCGTTCGCTA. Using differing reverse primers, PCR templates and hence carrier RNAs of differing length are produced.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>carrier</td>
			<td>length</td>
			<td>uncapped/&#181;g</td>
			<td>capped/&#181;g</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1034</td>
			<td>3.96</td>
			<td>0.45</td>
		</tr>
		<tr>
			<td>2</td>
			<td>966</td>
			<td>8.36</td>
			<td>0.95</td>
		</tr>
		<tr>
			<td>3</td>
			<td>889</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>4</td>
			<td>821</td>
			<td>6.6</td>
			<td>0.75</td>
		</tr>
		<tr>
			<td>5</td>
			<td>744</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>6</td>
			<td>676</td>
			<td>3.08</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>7</td>
			<td>599</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>8</td>
			<td>531</td>
			<td>3.96</td>
			<td>0.45</td>
		</tr>
		<tr>
			<td>9</td>
			<td>454</td>
			<td>2.64</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>386</td>
			<td>2.2</td>
			<td>0.25</td>
		</tr>
	</tbody>
</table>
Table 3: RNA carrier mix. In total 49 &#181;g of the carrier mix 0.3-1 kbp: uncapped = 44 &#181;g, capped = 5 &#181;g.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Total RNA input /ng</td>
			<td>PCR cycles</td>
		</tr>
		<tr>
			<td>1 ng</td>
			<td>18</td>
		</tr>
		<tr>
			<td>2 ng</td>
			<td>17</td>
		</tr>
		<tr>
			<td>5 ng</td>
			<td>16</td>
		</tr>
		<tr>
			<td>10 ng</td>
			<td>15-16</td>
		</tr>
		<tr>
			<td>25 ng</td>
			<td>14-15</td>
		</tr>
		<tr>
			<td>50 ng</td>
			<td>13-15</td>
		</tr>
		<tr>
			<td>100 ng</td>
			<td>12-14</td>
		</tr>
	</tbody>
</table>
Table 4: Expected number of PCR cycles in dependence of sample total RNA input. Approximate number of cycles is based on experiments performed using Saccharomyces cerevisiae, Drosophila melanogaster, and Mus musculus total RNA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Total RNA input/ng</td>
			<td>% overall mapped</td>
			<td>% uniquely mapped</td>
			<td>% carrier</td>
		</tr>
		<tr>
			<td>1 ng</td>
			<td>30</td>
			<td>20-30</td>
			<td>30</td>
		</tr>
		<tr>
			<td>2 ng</td>
			<td>60</td>
			<td>20-50</td>
			<td>10</td>
		</tr>
		<tr>
			<td>5 ng</td>
			<td>60-70</td>
			<td>40-60</td>
			<td>5-10</td>
		</tr>
		<tr>
			<td>10 ng</td>
			<td>60-70</td>
			<td>40-60</td>
			<td>5-10</td>
		</tr>
		<tr>
			<td>25 ng</td>
			<td>65-80</td>
			<td>40-70</td>
			<td>0-5</td>
		</tr>
		<tr>
			<td>50 ng</td>
			<td>65-80</td>
			<td>40-70</td>
			<td>0-3</td>
		</tr>
		<tr>
			<td>100 ng</td>
			<td>70-85</td>
			<td>40-70</td>
			<td>0-2</td>
		</tr>
	</tbody>
</table>
Table 5: Expected mapping efficiency and in dependence of total RNA input amount. Approximate numbers are presented and based on experiments performed using Saccharomyces cerevisiae and Mus musculus total RNA.
Supplementary Table 1: Primer sequences. <a href="https://www.jove.com/files/ftp_upload/59805/supptable1.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 2: Annealing of 5&#8217; and 3&#8217; linkers. <a href="https://www.jove.com/files/ftp_upload/59805/supptable2.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 3: 5&#8217; linker annealing. <a href="https://www.jove.com/files/ftp_upload/59805/supptable3.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 4: 5&#8217; linker mixing. <a href="https://www.jove.com/files/ftp_upload/59805/supptable4.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 5: 5&#8217; linker dilution. <a href="https://www.jove.com/files/ftp_upload/59805/supptable5.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 6: 3&#8217; linker annealing. <a href="https://www.jove.com/files/ftp_upload/59805/supptable6.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 7: 3&#8217; linker dilution. <a href="https://www.jove.com/files/ftp_upload/59805/supptable7.pdf" target="_blank">Please click here to view this table (Right click to download).</a>
Supplementary Table 8: Preparation of standard serial dilutions. <a href="https://www.jove.com/files/ftp_upload/59805/supptable8.pdf" target="_blank">Please click here to view this table (Right click to download).</a>

Watch this Scientific Journal Video about Transcription Start Site Mapping Using Super-low Input Carrier-CAGE at JoVE.com

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5&#8217;ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). ...

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