GSM9284916: HEK293T, 1X-Cys, biol rep 2; Homo sapiens; RNA-Seq
Cells were lysed using 1 mL of TRIzol™ (Thermo Fisher Scientific) and the samples were stored at -80 °C overnight. Subsequently, 200 μL of chloroform was added and the RNA phase was collected following centrifugation for 10 minutes at 4°C at 12700 rpm. The RNA fraction was precipitated with 500 μL of 2-propanol and washed with 1 ml of EtOH 70%. Finally, the pellet was left to dry and resuspend in 25 μL of RNAse-free water. Bulk mRNA sequencing libraries were prepared with 1ug of total RNA of each sample using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, NEB) with NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490, NEB), according to standard manufacturer's protocol. Total RNA and the final library quality controls were performed using Qubit™ Flex Fluorometer (Q33327, Thermo Fisher Scientific) and 4200 TapeStation System (G2991BA, Agilent) before and after library preparation. The libraries were sequenced on Element AVITI (2 x 75 bp, on average 20M reads per sample) and demultiplexed by bases2fastq software (Element).