GSM7864740: L; Homo sapiens; RNA-Seq
cells were washed 1X with 10 mL of 1X PBS and incubated with 5 mL of TrypLETM Express Enzyme (12604013, Thermo Fisher Scientific) at 5% CO2 and 37°C. After cells were fully detached, 5 mL of DMEM were added to each flask, and cells were collected into 15 mL tubes and centrifuged for 5 min at 0.3g. Cell pellets were resuspended in 1 mL of 1X PBS and centrifuged once again for 5 min at 0.3g. Finally, cell pellets were resuspended in 300 μL of TRIzolTM Reagent (15596026, Life Technologies) and incubated for 5 min. 300 μL of 100% ethanol was added to each tube and total RNA was extracted with Direct-zol RNA Miniprep Plus kit (R2070, Zymo Research). 20 ng of RNA per sample were subjected to Illumina TruSeq Stranded mRNA Library Prep Kit (20020594, Illumina) and TruSeq RNA Single Indexes Set A (20020492, Illumina) for RNA-seq on the Illumina NextSeq500 using the NextSeq 500/550 High Output Kit v2.5 (75Cycles) (20024906, Illumina).