GSM8940310: Homo sapiens, mesenchymal stem/stromal cells, control, biol repl3; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RNA quantity and purity were assessed with a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and integrity was confirmed using a Bioanalyzer 2100 (Agilent, CA, USA; RIN > 7.0) and denaturing agarose gel electrophoresis. Poly(A) mRNA was isolated from 1 μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) with two purification rounds, fragmented at 94°C for 5–7 minutes using the Magnesium RNA Fragmentation Module (NEB, cat. E6150, USA), and reverse-transcribed into cDNA with SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA). Second-strand DNA was synthesized using E. coli DNA polymerase I (NEB, cat. M0209, USA), RNase H (NEB, cat. M0297, USA), and dUTP Solution (Thermo Fisher, cat. R0133, USA). After adding A-bases to the blunt ends, indexed adapters were ligated, and size selection was performed using AMPureXP beads. The U-labeled second-strand DNA was treated with UDG enzyme (NEB, cat. M0280, USA), followed by PCR amplification (initial denaturation at 95°C for 3 min; 8 cycles of 98°C for 15 s, 60°C for 15 s, 72°C for 30 s; final extension at 72°C for 5 min). The resulting cDNA libraries (average insert size: 300 ± 50 bp) were sequenced on an Illumina NovaSeq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) using 2×150 bp paired-end sequencing (PE150).