GSM9455548: RNA-seq, Sia2,6hiPD-1−CD8+ T, replication 3; Mus musculus; RNA-Seq
CD8+T subsets were subjected to total RNA extraction. Total RNAs was extracted from FACS-isolated cells by using TRIzolTM (Thermo) following the manufacturers' instructions. For input cells less than 100,000, the aqueous phase of the TRIZol/chloroform extraction was carefully transferred into a new tube and the RNAs were cleaned and concentrated by using the ZYMO RNA Clean & Concentrator following the manufacturers' instructions. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library was sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.