GSM8767538: mESC Tet1/2/3 CTRL, Input, PyBo, clone4, rep2; 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. Ribosomal RNA was depleted from samples using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB) following the manufacturer's protocol. RNA was T4 PNK (NEB) treated before library construction. FIBo-seq library construction was performed with the NEXTFLEX Small RNA-Seq Kit V4 with UDIs (PerkinElmer) using the NEXTFLEX small RNAseq PreArrayed UDI Barcode primers. Libraries were prepared with a starting volume of 4 µl for the f5C-RIP samples (~ 0.823 ng) and a starting amount of 0.823 ng RNA for the input samples following Step A to Step F of Perkin Elmer`s standard protocol (V23.04). Step A (NEXTFLEX 3` 4N Adenylated Adapter Ligation) was performed overnight at 16°C and Step F (PCR amplification) with 24 PCR cycles. Thereafter, the no size selection option in the protocol was followed. Libraries were profiled in a High Sensitivity DNA Chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies) and pooled together in equimolar ratio. The pool was purified by running a 3% agarose gel cassette on a PippinHT (Sage Science) and size-selected for library fragments in 175-250 bp size range (adapter size + insert size).
GEO
GSM8767538_r1