GSM9216758: IVT_RNA, mock, rep1; Mus musculus; RNA-Seq
Total RNA was isolated from cultured cells using QIAzol Lysis Reagent (Qiagen) according to the manufacturer's protocol followed by TURBO DNase (Invitrogen) treatment and another round of QIAzol purification. Poly(A)+ RNA was purified from total RNA using Oligo d(T)25 Magnetic Beads (Thermo Fisher Scientific), following the manufacturer's protocol, with three consecutive rounds of selection. IVT RNA generation: 400 ng poly(A)+ RNA isolated from mESCs was incubated with 1.67 µM oligo(dT)-VN RT primer and 1.67 mM dNTPs for 5 min at 75°C. For RT reaction, template switching RT enzyme mix and buffer (NEB) and 3.75 µM template switching oligo was added and incubated for 90 min at 42°C and 5 min at 85°C. 2nd strand cDNA synthesis was performed with Q5 Hot Start High-Fidelity DNA Polymerase (NEB), RNase H and 0.2 µM T7 extension primer for 15 min at 37°C, 1 min at 95°C and 15 min at 65°C. Double-stranded DNA was purified with HighPrep PCR beads (0.8x beads, MagBio) and used for in vitro transcription with the HiScribe T7 High Yield RNA Synthesis Kit (NEB) and canonical NTPs. Synthesized RNA was purified using DNase I (NEB) and the RNA Clean & Concentrator kit (Zymo research) following the manufacturer's protocol. IVT RNA was size selected with HighPrep RNA Elite beads (0.6x beads, MagBio). PyBo treatment: IVT RNA was incubated in 900 mM pyridine borane in 600 mM sodium acetate (pH 5.2) at 37°C for 24 hours. For mock treated samples, water was added instead of chemicals. RNA was purified using the RNA Clean & Concentrator kit (Zymo research). RNA library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (20020594, Illumina), entering the protocol at the RNA fragmentation step and excluding the poly(A) enrichment. 20 ng RNA was used as starting amount. Libraries were size-selected for >100 bp fragments.