GSM9825249: dHL-60 cells, control sgRNA,  rep2; Homo sapiens; RNA-Seq
Total RNA was isolated and purified using TRIzol reagent and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0. Poly (A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher Scientific) for two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, e6150) under 94 °C for 5-7 min. Then the cleaved RNA fragments were reverse transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific, 1896649). These were then used to synthesize U-labeled second-stranded DNA with E. coli DNA polymerase I (NEB, m0209), RNase H (NEB, m0297) and dUTP Solution (Thermo Fisher Scientific, R0133). Single or dual index adapters were ligated to the fragments. After heat-labile UDG enzyme (NEB, m0280) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR in the following conditions: initial denaturation at 95 °C for 3 min, 8 cycles of denaturation at 98 °C for 15 sec, annealing at 60 °C for 15 sec, and extension at 72 °C for 30 sec, and then final extension at 72 °C for 5 min. The average insert size for the final cDNA library was 300 ± 50 bp.