The dFab_CCR-IL2 positive and nontransduced human T cells from 3 individual donors were isolated using CD34 microbeads (Miltenyi Biotec, 130–046–702) and rested for 24 hours in R10 media. Subsequently, the T cells were seeded at 1 × 106 cells/mL with or without 100,000 iU/mL of human IL2. After 72 hours, rHuIL2 was replenished. After 7 days, the cells were lysed, and the mRNA was extracted with the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare, 12183018A). The RNA was quantified with NanoDrop (Thermo Fisher Scientific). The quality of the RNA was assessed with Agilent TapeStation (Agilent, RRID:SCR_019547). Poly-A selection was performed to remove ribosomal RNA. Roughly, 40 million paired end reads (150 bp) were sequenced on Illumina Hiseq sequencer (GeneWiz) for each individual CAR T-cell sample with different CCR modules.
Fastq reads were mapped to the human genome (B37.3) and gene model GenCode (V19) with Omicsoft Aligner 4 (18 (link)). Gene count data was normalized by logGenometric mean values. Differential gene expression analysis was carried out using DeSeq2 General Linear Model test (RRID:SCR_015687; ref. 19 (link)). Gene set enrichment analysis (Subramanian) Pre-ranked tool (GSEAPreranked) was used to conduct the pathway and functional analysis on the differentially expressed genes.
Fastq reads were mapped to the human genome (B37.3) and gene model GenCode (V19) with Omicsoft Aligner 4 (18 (link)). Gene count data was normalized by logGenometric mean values. Differential gene expression analysis was carried out using DeSeq2 General Linear Model test (RRID:SCR_015687; ref. 19 (link)). Gene set enrichment analysis (Subramanian) Pre-ranked tool (GSEAPreranked) was used to conduct the pathway and functional analysis on the differentially expressed genes.
