Instrument: Illumina HiSeq X Ten
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: For rRNA(-) nuclear RNA-seq, cell fractionation was performed, and the resulting RNA was subjected to two rounds of rRNA removal using RiboMinus™ Eukaryote Kit for RNA-Seq .For polyA RNA-seq, total RNA was isolated from cell lines with Trizol (invitrogen) according to the manufacturer's instructions. Two rounds of poly-A selection were performed using Dynabeads™ Oligo(dT)25 (Invitrogen) to obtain mRNA. For PAR-CLIP-seq, five 15 cm dishes of HEK293T cells were transfected with flag-tagged PUS10 plasmid at 80% confluency. After 6 h, the media was changed and 200 μM 4SU was added. After additional 18 h, the cells were washed once with 10 ml ice-cold PBS for each plate. Then, the plates were kept on ice, and the UV crosslink was carried out twice by 150 mJ/cm2 at 365 nm. After immunoprecipitation, the final recovered RNA sample was further cleaned by RNA Clean & Concentrator (Zymo Research). For DM-Ψ-seq, small RNA (<200 nt) was isolated and purified from cell culture using miRNeasy Mini Kit, RNeasy MinElute Cleanup Kit and RNase-free DNase Set (Qiagen) according to the manufacturer's instructions. The rRNA(-) nuclear RNA and poly-A RNA were used to construct RNA-seq library under the instruction of NEBNext® Ultra™ RNA Library Prep Kit for Illumina. For PAR-CLIP-seq, the RNA samples were used to construct library under the instruction of NEBNext® Multiplex Small RNA Library Prep Set for Illumina. For DM-Ψ-seq, the library construction was performed according to the eCLIP library construction protocol with several modifications.