title: GSM6199064: Donor2_CAR_Stim4 [FB4E_S104_L004_R1_001]; Homo sapiens; RNA-Seq
library_construction_protocol: T cells were subjected to the repeated stimulation assay in 24-well plates, transferring T cells to freshly seeded cancer cells every 48 hours. Cultured T cells were sorted after each stimulation using BD FACSAria Fusion to obtain a pure population of NY-ESO-1 multimer positive cells T cells from the co-cultures with target cancer cells, and resuspended in TRI Reagent (Sigma, Cat.# T9424). Total RNA was extracted using Direct-zol RNA MicroPrep kit (Zymo research, Cat.# R2061) For paired-end samples, RNA-seq libraries were prepared by Oligo dT enrichment followed by a stranded Illumina library prep protocol with the KAPA mRNA HyperPrep kit (Kapa Biosystems, KK8580). Libraries were checked for quality on an AATI Fragment analyzer (Agilent, DNF-935–1000), quantified using the Illumina Quant Universal qPCR Mix (Kapa Biosystems, KK4824), and pooled evenly at 3nM. For single read samples (RASA2KO and CTRL Stim5), batch-Tag-seq protocol was used (PMID 27037501).