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.