title: GSM8900648: control _S10; Mus musculus; RNA-Seq
library_construction_protocol: CD8 T cells were collected and  stored in RLT buffer (Qiagen) supplemented with 1% vol/vol β-mercaptoethanol. RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad) according  to manufacturer's instructions. RNA Sequencing Library Generation RNA was assessed for quality using an Agilent TapeStation 4150/Fragment Analyzer 5300 and RNA concentration was quantified on a Qubit FLEX fluorometer. Libraries were generated with the Illumina Stranded mRNA Library Prep kit (Illumina: 20040534) according to the manufacturer's instructions. Briefly, 50 ng of input RNA was used for each sample. Following adapter ligation, 14 cycles of indexing PCR were completed, Illumina RNA UD Indexes. Library quantification and assessment was done using a Qubit FLEX fluorometer and an Agilent TapeStation 4150/Fragment Analyzer 5300. Libraries were normalized and pooled to 2 nM by calculating the concentration based off the fragment size (base pairs) and the concentration (ng/l) of the libraries.  Library Sequencing Sequencing was performed on an Illumina NextSeq 2000, using a P4 100 flow cell. The pooled library was loaded at 750 pM and sequencing was carried out with read lengths of 2x58 bp, with a target of 40 million reads per sample. Sequencing data was demultiplexed by the on-board Illumina DRAGEN FASTQ Generation software (v3.10.12).