GSM9853622: CD4, BCAN synNotch - anti-EphA2/IL13RA2 CAR, donor 2V, replicate 3; Homo sapiens; RNA-Seq
Total RNA was extracted from CD4+ and CD8+ T-cells transduced with either Constitutive CAR or B-SYNC using the RNeasy Mini Kit (Qiagen, 74106) with on-column DNase treatment (RNase-Free DNase Set, Qiagen, 79254) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent Bioanalyzer 5400 and confirmed to have a RIN (RNA Integrity Number) value of 7 or greater (range: 7–9.4). RNA quality assessment, library preparation, and sequencing were performed by Novogene (Sacramento, CA). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand cDNA synthesis. Libraries were constructed through end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Quality control was conducted using Qubit and real-time PCR for quantification, and Bioanalyzer for size distribution assessment.