GSM6886674: EphA2 CAR transduced T cell co-cultured with U251; Homo sapiens; RNA-Seq
Total RNA was extracted from cell using Trizol Reagent (Invitrogen) following the manufacturer's instructions. The quantity and purity of RNA were measured on a Nanodrop Technologies ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). Only samples with the appropriate absorbance measurements (A260/A280 of ~2.0, and A260/A230 of 1.9-2.2) were considered for inclusion in the present study A total amount of 2 µg total RNA per sample was used as input material for generating the sequencing libraries using NEBNext®Ultra™ RNA Library Prep Kit (#E7530L, NEB, USA) following the manufacturer's. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, A-tailing and adapter added were implemented. The aimed products were retrieved and PCR was performed, then the library was completed.