title: GSM6133761: ALKBH11ko, ip, 1; Homo sapiens; RIP-Seq
library_construction_protocol: TotalRNA extracted from WT and ALKBH1 knock out cells were first treated with 2 μl DNase I (NEB) to remove DNA contamination. The total RNA was then fragmented into 150 nt fragments using RNA Fragmentation Reagents (Thermo). 100 ng f5C containing oligo was first added into 100 μg fragmented RNA, the mix was then incubated with 2 mM ARP in a 40 mM aqueous NH4OAc buffer pH 5.0 supplemented with 100 mM anisidine for 24 h at 25°C. RNA was then purified using phenol/chloroform extraction. 200 ng RNA was taken out as input, the rest of the samples were incubated with 50 μl pre-washed streptavidin beads in 400 μl IP -buffer (200 mM NaCl, 0.4 mM EDTA, 50 mM PH=7.4 Tris-HCl pH 7.4, and 0.1% NP-40) for 2 hours at 4 °C. The beads were then washed twice with IP -wash buffer (500 mM NaCl, 0.4 mM EDTA, 50mM PH=7.4 Tris-HCl pH 7.4, and 0.1% NP-40). The beads and input samples were treated with Quick-CIP (NEB) at 37 °C for 30 mins with rotation, the input samples were purified using RNA Zymo Clean kit (Zymo Research), and the beads were washed twice with wash buffer. Reverse transcription was performed using oligonucleotides containing randomized barcodes (UMIs) and two inversely oriented adaptor regions separated by a BamHI restriction site. cDNAs were size-purified on TBE-Urea gels and then circularized by CircLigase II (Epicentre). Circularized cDNAs were digested with BamHI and linearized cDNAs were amplified using Solexa primers and submitted for Illumina sequencing. Iclip-seq