GSM7429945: OT-I T cells, vehicle treated, 24h, replicate2; Mus musculus; RNA-Seq
RNA extraction was performed usdingh the Arcturus™ PicoPure™ RNA Isolation Kit (KIT0204, ThermoFisher Scientific). Traces of genomic DNA were removed using RNase-Free DNase Set (79254, QIAGEN) following the on-column digesting protocol of the RNA isolation kit. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and  index codes were added to attribute sequences to each sample. 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 NEBNext 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, NEBNext Adaptor with hairpin loop structure were ligated to  prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp  in length, the library fragments were purified with AMPure XP system (Beckman Coulter,  Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated  cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. 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.