GSM8769179: mESC Tet1/2/3 TKO, RIP, mock, clone 26; Mus musculus; RIP-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. RNA was chemically fragmented to 120-180 nt-long fragments followed by immunoprecipitation with an anti-f5C-antibody (1 µg anti-f5C-antibody/ 5 µg RNA; #61227, Active Motif). Samples were 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 pyridine borane. RNA was T4 PNK (NEB) treated before library construction. Library construction 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.