GSM8769163: mESC Alkbh1 KO, PyBo, clone3; 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. Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit v2 (human/Mouse/Rat) (NEB) following the manufacturer's protocol and purified using the RNA Clean & Concentrator kit (Zymo Research). For PyBo-seq, 200 ng of RNA was incubated in 900 mM pyridine borane in 600 mM sodium acetate (pH 5.2) at 37°C for 24 hours. For Mal-seq, 200 ng of RNA was incubated in 150 mM malononitrile in 1x TE buffer (pH 7.4) at 37°C for 24 hours. For mock treated samples, water was added instead of chemicals. For PyBo-seq of ribosomal RNA, total RNA was chemically fragmented to 120-180 nt-long fragments and PyBo-treated as described above. 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. 10 ng RNA was used as starting amount. Libraries were size-selected for >100 bp fragments. Library construction of rRNA was done with the NEXTFLEX Small RNA-Seq Kit V3 (Bioo Scientific) following Step A to Step G of Bioo Scientific`s standard protocol (V19.01) using the NEXTFLEX 3' SR Adaptor and 5' SR Adaptor. Step A (NEXTFLEX 3` 4N Adenylated Adapter Ligation) was performed overnight at 20°C and for step F (Bead Cleanup) the modified protocol was used to retain small RNAs. Libraries were prepared with a starting amount of 1.4 ng RNA and amplified in 21 PCR cycles. Libraries were size-selected for 18 – 362 nt insert size.