title: PROseq_d0-3 (CRX1229830; CRA019813)
library_construction_protocol: 1x10e7 naive or exhausted CD8+ T cells were harvested and permeabilized twice in perm buffer. Then the run-on reaction was conducted at 37 degree for 5 minutes in the presence of 50 uM bitoin-11-CTP and bitoin-11-UTP (Perkin Elmer) together with 125 uM ATP and GTP (Sigma). Total RNAs were extracted by TRIzol LS (Thermo Fisher) and fragmented by NaOH hydrolysis on ice for 15 minutes. The biotin labeled RNAs were purified by Streptavidin C1 beads (Thermo Fisher) and eluted by TRIzol. 3'RNA adaptor was ligated to biotin labeled RNA at 25 degree for 2.5 hours. Then the ligated RNAs were further purified by Streptavidin C1 beads. Then ligated RNAs were treated by T4 PNK (NEB) and RppH (NEB) on beads. Then 5'RNA adaptor was ligated to the biotin labeled RNA at 25 degree for 2.5 hours on beads. After ligation, RNAs were purified by TRIzol extraction and reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher) with RT primer. The reverse transcription reaction mixture was directly PCR amplified using Ultra II Q5 mix (NEB) with indexing primers for 15 cycles. Finally, library was size-selected using 2.5% Low Melting Point agarose gel (Thermo Fisher) with a range of 160-400 bp. The library was sequenced on an Illumina NovaSeq 6000 platform (Illumina).
identifiers.namespace: Beijing Institute of Genomics, Chinese Academy of Sciences
identifiers.id: DRX719552
identifiers.namespace: NGDC
identifiers.id: CRX1229830