Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases
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: ReCappable-seq procedureTotal RNA was (optionally, see text) de-phosphorylated using the Quick CIP (NEB M0525) (3 units/ µgRNA at 0.6 units/µL) and the resulting RNA was purified with the “Clean and concentrate” kit (Zymo Research R1013) using the standard protocol.
Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro, RNA Sequencing Assay