GSM8851896: BM LSCs, CD37 WT, rep1; Mus musculus; RNA-Seq
Total RNA was purified from cells using TRIzol reagent (TransGen Biotech). 1 ug of total RNA was used for the construction of sequencing libraries. The purity and concentration of each RNA sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). RNA integrity was evaluated by Bioanalyzer 2100 (Agilent Technologies, California, USA). Poly(A) RNA was purified from total RNA using Dynabeads Oligo (dT) 25-61005 (Thermo Fisher Scientific) and fragmented into small pieces at 94°C for 5–7 minutes using the Magnesium RNA Fragmentation Module (NEB, e6150). Escherichia coli DNA polymerase I (NEB, m0209), RNase H (NEB, m0297), and dUTP solution (Thermo Fisher Scientific, R0133) were used for cDNA synthesis. Next, A base was added to the blunt end of each strand to prepare for ligation to the indexed adapter. Each adapter contains a T base overhang, which is used to ligate the adapter to the A-tailed fragmented DNA. The single-index or dual-index adapter was connected to the fragment and the size selection was performed using AMPureXP beads. After treating the U-labeled second-strand DNA with UDG enzyme (NEB, m0280), the ligated product was amplified by PCR under the following conditions: initial denaturation at 95°C for 3 minutes; 8 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds; followed by extension at 72°C for 5 minutes. The average insert size of the final cDNA library was 300 ± 50 bp.