HT-5Pseq and 5’-Capseq protocols
HT-5Pseq libraries were prepared as previously reported (Zhang & Pelechano, 2021). Briefly, 6 μg of DNA-free total RNA were directly ligated with the RNA/RNA oligo containing UMI (RNA rP5_RND oligo). Ligated RNA was reverse-transcribed and primed with Illumina PE2 compatible oligos containing random hexamers and oligo-dT. RNA in RNA/DNA hybrid was depleted by sodium hydroxide with 20-minute incubation at 65°C. Ribosomal RNAs were depleted using DSN (duplex-specific nuclease) with the mixture of ribosomal DNA probes. Samples were amplified by PCR and sequenced in an Illumina NextSeq 500 instrument using 60 sequencing cycles for read1 and 15 cycles for read 2.
For 5’Capseq, 5’capped mRNAs were captured as previously described (Pelechano et al., 2016). Specifically, 10 µg total RNA were treated with calf intestinal alkaline phosphatase (NEB, CIP) to remove 5′P molecules (fragmented and non-capped). After purification, mRNA 5’caps were removed by Cap-Clip (Biozyme), which resulted in 5’P molecules from previously capped molecules. The following steps were the same as that described for HT-5Pseq (see above) with variations only for skipping removing ribosomal RNA. Datasets are available from GEO database: GSE119114 for the 5Cap-seq data, GSE193992 (reviewer access code:ahadwoocjrivtot ) for HT-5Pseq data.