GSM8911782: MSC(M), Direct FBS, 1 Passage, Donor 2; Homo sapiens; RNA-Seq
Total RNA was extracted using a High Pure RNA Isolation Kit (11828665001, Roche) according to the manufacturer's instructions. Briefly, cells were resuspended in PBS and lysed by adding Lysis/-Binding Buffer, followed by centrifugation through a High Pure Filter Tube. After lysis, DNA contamination was removed by DNase I treatment, and subsequent washing steps were performed to increase purity. The isolated RNA was eluted with Elution Buffer and stored at -80ºC until further use. Library preparation and paired-end 150 bp sequencing were performed by Novogene (Beijing, China) using an Illumina NovaSeq 6000 platform. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the directional library, it was ready after end repair, A tailing, adapter ligation, size selection, USER enzyme digestion , amplification, and purification. The library was checked with Qubit and real time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.