title: GSM5980024: BL3POS; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized by intraperitoneal injection of sodium pentobarbital and then intracardiacally perfused with 30 ml of Phosphate Buffer Saline (Corning). Blood were collected in PBS with 0.5 mM of Ethylenediaminetetraacetic acid (EDTA, Boston BioProducts, Inc.). Red blood cells were lysed in 5 ml of RBC Lysis Buffer (Affymetrix eBioscience). 500 CD44+ CD62L- PD-1+ and PD-1- CD8 T cells from the blood were sorted directly into nuclease free water (final volume = 9.5 l) and processed by the NGS Platform of the Center for Advanced Genomics Technology at ISMMS. Cell samples were passed to whole-transcriptome library preparation using the Takara Bio SMART-Seq v4 PLUS Kit following the manufacturer's instructions. Briefly, cDNA synthesis from low amounts of RNA was enabled through Takara Bio's SMART technology. 3' ends of cDNA were then adenylated prior to ligation with adapters utilizing unique dual indices (96 UDIs) to barcode samples to allow for efficient pooling and high throughput sequencing. Libraries were enriched with PCR, with all samples undergoing 16 cycles of amplification prior to purification and pooling for sequencing. Completed libraries were quantified using Quant-iT reagents and equimolar pools were generated and sequenced on the NextSeq 550 using a high output 150 cycle flow cell generating paired end 75 bp reads and approximately 30 million reads per sample.