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).

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	<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. 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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, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>59304-100ML-F</td><td>Used in RNAclean XP purification.</td></tr><tr><td>3&#039; 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&#039;linkers is described in the supplementary of this protocol.</td></tr><tr><td>5&#039; 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&#039;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&#039; and 3&#039;-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, &amp;ge;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 (&lt;em&gt;E. coli&lt;/em&gt;)</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 &amp;micro;L, 1-20 &amp;micro;L, 20-200 &amp;micro;L)</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 &amp;mu;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 1,000 &amp;mu;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 &amp;mu;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 &amp;mu;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, 1 M 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&#039;linker&#039;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). ...

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

Research

JoVE Journal

Genetics

12.0K Views.  Imperial College London. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van genregulatie, omdat het kernpromotor en versterkerdetectie mogelijk maakt met behulp van lage hoeveelheden uitgangsmateriaal. Het belangrijkste voordeel van deze techniek is dat het onbevooroordeelde, genoom-brede enkele nucleotide resolutie mapping van RNA polymerase II transcriptie start sites met behulp van slechts 10 nanogram van de totale RNA. CAGE blijkt van onschatbare waarde voor het blootleggen van ...

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

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Biology

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 ...

Bioengineering

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

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

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