GSM5101168: OT-I RNA-seq 60 min. N4 peptide rep2; Mus musculus; RNA-Seq
Samples of stimulated CD45.2+ OT-I cells and CD45.1+ wildtype splenocytes mixtures were stained with CD8a and CD45.2 antibodies and 7-AAD and OT-I cells were sorted (BD FACSAria) into 100% FBS and pelleted. Total RNA from ~300,000 OT-I cells per sample was collected with the RNeasy micro kit (Qiagen) with a 15-minute on-column DNase digestion (Qiagen) to remove genomic DNA. Total RNA quality and quantity was determined with fragment analysis (University of Massachusetts Medical School Molecular Biology Core Lab) and Qubit Fluorometer (Invitrogen) analysis. cDNA libraries were generated following a modified paired-end SMART-seq protocol (Kriegsman et al., 2019; Ramsköld et al., 2012). At least 20 ng of input RNA was used for reverse transcription with SMARTscribe reverse transcriptase (Clontech). Whole transcriptome amplification (WTA) was performed with Advantage 2 polymerase (Takara Bio). WTA reactions were monitored with qPCR to determine optimal cycle number. WTA libraries were then sized-selected with AMPure XP DNA SPRI beads (Beckman Coulter), tagmented with Tn5 transposases (Illumina Nextera XT), barcoded, and amplified with cycle number determined via qPCR monitoring. Final libraries were further size-selected with SPRI beads to an average size of 300-500 bp and quality was assessed with fragment analysis and Qubit analysis. Libraries were pooled and sequenced on a NextSeq 500 sequencer (Illumina).