Journal: Genome Research

Article Title: Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

doi: 10.1101/gr.275784.121

Figure Lengend Snippet: ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

Article Snippet: Capping with 3′ desthiobiotin GTP (DTB-GTP) was performed in 50 µL total volume with 5 µL VCE (NEB M02080) and 0.5 mM DTB-GTP (NEB N0761), in the absence of SAM for 40 min at 37°C.

Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro

Journal: bioRxiv

Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

doi: 10.1101/696559

Figure Lengend Snippet: ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

Article Snippet: Capping with 3’ Desthiobiotin GTP (DTB-GTP) was performed in 50 µl total volume with 5 µL Vaccinia capping enzyme (NEB M02080) and 0.5 mM DTB-GTP (NEB N0761), in the absence of SAM for 40 min at 37°C.

Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro, RNA Sequencing Assay