GSM6604728: HeLa_IMP1_iCLIP_tech_replicate_2; Homo sapiens; OTHER
iCLIP was performed using a previously reported protocol48 (see also Supplementary Figure 11a) which was adapted as described below. Briefly, three HeLa cell biological replicates in 10 cm dish (80% confluency) were cross-linked at 150 mJ/cm2 in a Stratalinker 2400 at 254 nm and then lysed in 1 mL of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630 (Sigma I8896), 0.1% SDS, 0.5% sodium deoxycholate. 0.8 U of RNase I (Thermo Scientific, EN0602) and 4 μL of Turbo DNase I (Ambion, AM223) were added to 1 mg (protein content) of lysate for RNA fragmentation and DNA digestion respectively. Samples were incubated with 5 μg of anti-IMP1 (Rabbit anti-IMP1 (MBL, RN007P) or 5 μg of anti-IgG (Proteintech 30000-0-AP) antibodies, and coupled to Protein G beads (Dynal), rotating on a wheel at 4°C overnight. Pre-adenylated infrared dye-labelled IRL3 adaptor (IRdye-800CW-DBCO (LI-COR, cat# 929-50000)49 with sequence /5rApp/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA /iAzideN/AA AAA AAA AAA A/3Bio/ was ligated to RNA. The protein-RNA complexes were then separated by SDS-PAGE29, blotted onto nitrocellulose membrane and visualised with an Odyssey scanning system (LI-COR). The desired region (determined from the RNAse gradient experiment in S11c) was excised from the membrane in small pieces and RNA was released using proteinase K (Roche, 03115828001) digestion and incubation at 60 min at 50°C. For RNase gradient experiment, 0.4, 0.8 or 2.5 U of RNase I (Thermo Scientific, EN0602) and 4 μL of Turbo DNase I (Ambion, AM223) were added to 1 mg (protein content) of lysate for RNA. Phenol-Chloroform precipitation was performed to recover RNA. Reverse transcription was performed using Superscript IV Reverse Transcriptase (Life Technologies), using primers containing Unique Molecular Identifier barcodes to allow multiplexing. cDNA molecules were purified using AMPure XP beads (Beckman-Coulter, USA), circularised using Circligase II (Epicenter), and purified using AMPure XP beads (Beckman). After PCR amplification, libraries were size-selected by gel-purification and size distribution was assessed using a2400 Bioanalyser (Agilent). QuBit dsDNA HS Assay (ThermoFisher Scientifics) was used to quantify libraries. The same quantity of cDNA for each sample in the library was sequenced as single end100bp reads on Illumina HiSeq 4000.