GSM5776018: NEC + ST266 oral 2; Mus musculus; RNA-Seq
Total RNA was purified and DNAse I treated using RNeasy mini kit (Qiagen) The total RNA quantity and purity were analyzed using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511), and high-quality RNA samples with RIN number > 7.0 were used to construct sequencing libraries. After total RNA was extracted, mRNA was purified from total RNA (5μg) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat # E6150, USA) at 94℃ for 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, cat # 1896649, USA), which were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat # M0297, USA) and dUTP Solution (Thermo Fisher, cat # R0133, USA). 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. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After heat-labile UDG enzyme (NEB, cat. # M0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR in 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 libraries was 300 ± 50 bp.