GSM4861496: R4 Continuous CAR+ T2 [RNA-seq]; Homo sapiens; RNA-Seq
For flow cytometry and sorting assays of CAE, M5 CAR T cells were stained in fluorescence-activated cell sorting (FACS) buffer consisting of PBS (Gibco), 0.5% bovine serum albumin (BSA) (GEMINI), 2 mM EDTA (Invitrogen), and 100ug/ml DNase (Roche). CountBrightTM Absolute Counting Beads, (ThermoFisher) were used as an internal standard according to the manufacturer's instructions to calculate absolute cell counts in cell suspensions.  Antibodies specific for human CD45 (ref 304032, clone HI30), CD3 (317322, OKT3), CD4 (357412, A161A1 / 317440), CD56 (304608, MEM-188), EpCAM (324226/324238, 9C4), CD94 (305520, DX22), KLRB1 (339918, HP-3G10), TIGIT (372716, A15153G), TCR Va24-Ja18 (342922, 6B11), PD-1 (329928, EH12.2H7) were purchased from BioLegend. Antibodies specific for human CD8 (560179, SK1) was purchased from BD Pharmingen. Antibodies specific for human NKG2A (FAB1059P, 131411) and Methothelin (FAB32652P, 420411) were purchased from R&D Systems. Antibodies specific for human CTLA-4 (12-1529-42, 14D3) was purchased from eBioscience. Antibody specific for human NKG2C (130-103-636, REA205) was purchased from Miltenyi.  M5 CAR expression was assessed using biotinylated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, 109-066-006) followed by streptavidin (FITC or APC) (BioLegend) or using an anti-idiotype antibody provided by Novartis Pharmaceuticals. Live/dead staining was performed using a Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies following manufacturer's protocol followed by cell surface staining for 20 minutes at room temperature in the dark. Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher) according to the manufacturer's instructions. Samples were acquired on an LSRII Fortessa Cytometer (BD Bioscience) and analyzed with FlowJo v10 software (FlowJo, LLC). Sorting assays were performed using a FACS Aria Cytometer (BD Bioscience).    Total RNA was extracted using Qiazol (Qiagen) from 300 cells for day 0, day 16 or day 28 for CD8+ T cells continuously stimulated with antigen (two sorted populations including surface CAR-positive and surface CAR-negative cells). Cells were recovered by RNA Clean and Concentrator spin columns (Zymo), followed by incubation with oligo-dT. The transcription reaction was carried out on 100pg of cDNA for 1min at 55°C. RNA-seq libraries were made following the previously established SMARTseq2 protocol (Picelli et al., 2014). Libraries were uniquely barcoded (Buenrostro et al., 2013) and amplified for 14 cycles. Fragment size distribution was verified and paired-end sequencing (2 x 75 bp reads) was carried out on an Illumina NextSeq 500.