GSM6198371: WT_IMQ_3; Mus musculus; RNA-Seq
Total RNA was isolated and purified using TRIzol reagent (Invitrogen) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop 1000 (NanoDrop). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent) with RIN number > 7.0, and confirmed by electrophoresis with denaturing agarose gel. 1 μg of total RNA was used for the construction of sequencing libraries. Poly (A) RNA was purified from 1 μg total RNA using Dynabeads Oligo (dT) (Thermo Fisher) by two rounds of purification. Then the poly (A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module kit (NEB) at 94℃ for 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase (Invitrogen), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB), RNase H (NEB), and dUTP Solution (Thermo Fisher). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 ± 50 bp.