GSM5212192: HeLa_shINTS11_ctrl_r2_eCLIP_INTS11_IP; Homo sapiens; RIP-Seq
After double adapter trimming (cutadapt v1.14), resulting reads were first mapped against the repetitive genome using STAR (v2.7.6a), the unmapped output was aligned against the human genome (hg19). PCR duplicates were removed by umi_tools (v1.0.0) and the samples were visualized in UCSC. eCLIP was performed according to Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810 Libraries were prepared as described in Van Nostrand, E., Pratt, G., Shishkin, A. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–514 (2016). https://doi.org/10.1038/nmeth.3810. In brief, 2 x 10^7 HeLa cells were crosslinked by UV-C irradiation (254 nm, 400 mJ/cm2) and lysed on ice followed by sonication. The lysate was subjected to RNase I (Ambion) digest (40 U/ml) in presence of murine RNase inhibitor (NEB) and 4 U/ml Turbo DNase (Ambion). 4 µg INTS11 antibody (Sigma Prestige, HPA029025) was pre-incubated with Dynabeads M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11204D) for 1 hour and added to the lysates for immunoprecipitation at 4˚C for 16 hours. 2% of the lysate were removed and stored as size-matched input controls. Co-immunoprecipitated RNA was dephosphorylated, followed by on-bead 3'RNA adapter ligation using high concentration T4 RNA Ligase I (NEB). IP efficiency was verified by immunoblot of 20% of the IP samples. Input controls and 80% of the IPed INTS11-RNA complexes were run on a NuPAGETM 4-12% Bis-Tris Gel, transferred to nitrocellulose membrane and the desired size range (INTS11 signal + 75 kDa) was cut from the membrane for both input and IP samples. To extract RNA, nitrocellulose membranes were finely fragmented and treated with Urea/ Proteinase K, followed by acid phenol-chloroform extraction and purification using RNA Clean & Concentrator column cleanup (Zymo Research). Size-matched input samples were also dephosphorylated and ligated to 3'RNA adapter. After reverse transcription (AffinityScript reverse transcriptase, Agilent), excess oligonucleotides were removed with exonuclease (ExoSAP-IT, Affymetrix), and the remaining RNA hydrolyzed by NaOH. A 3' DNA Linker was ligated to the cDNA, and the resulting library was PCR amplified using Q5 Ultra IIMaster Mix (NEB). The library was size selected by agarose gel electrophoresis and purified (MinElute, Qiagen). Single-end sequencing was performed to an average of 40 million reads per sample using Illumina NovaSeq 6000.