GSM8459418: iCLIP hESCs, Dicer-S1344L hemizygous, Rep2; Homo sapiens; RIP-Seq
Dicer1 H1 hESCs of the following genotypes (wildtype, pm/fs, pm/pm) were expanded under dox-depleted conditions for 10 days. Two 15 cm dishes of confluent hESCs were used per sample. The cells were UV irradiated with 150mJ/cm2 UVC (254nM wavelength) two times. The cells were then lysed on ice with iCLIP lysis buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% SDC, protease inhibitor cocktail (Roche), and 40 U/ml RNaseOUT (Sigma)) for 20 min on ice. TurboDNase (Invitrogen #AM2238, 10U), RNase I (0.05 U/mL) were added subsequently, and samples were incubated at 37°C for 5 min. Treated lysates were immunoprecipitated with anti-Ago2 antibodies (Abnova) or control IgG (MilliporeSigma) coupled with Dynabeads protein G (Thermo Fisher Scientific) for 2 hrs at 4°C with gentle agitation. The immunoprecipitate was washed sequentially with high-stringency buffer, high-salt buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), and PNK wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, and 0.2% Tween-20). The RNAs in the immunoprecipitate were dephosphorylated on the beads with FastAP Thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and radiolabeled in 300 μl mixture: 60 μl 10X PNK buffer (NEB), 5 μl RNaseOUT, 2.0 μl γ-32P-ATP (15 μCi), 7 μl PNK enzyme (NEB M0201) and 222 μl H2O for 20 min at 37°C. The immunoprecipitated materials were washed with the high-salt buffer, and then 3' linker ligation was carried out 2 hrs at room temperature in 25 μl of mixture: 9 μl H2O, 9 μl 50% PEG-8000 (NEB #B1004), 3 μl 10x T4 RNA ligation buffer (NEB), 0.4 μl RNaseOUT, 0.3 μl ATP (100mM), 0.8 μl DMSO, 2.5 μl T4 RNA ligase 1 (10U/μl – NEB M0204). Protein–RNA complexes were eluted from the beads, electrophoresed on 4–20% Bis-Tris NuPAGE gels (Thermo Fisher Scientific), transferred to nitrocellulose membranes (Whatman), and visualized by autoradiography. The area of the membrane corresponding with proteins that were ~100 kDa (the MW of Ago2) to ~150 kDa in size was excised from each lane of the blot. Excised lanes were treated with proteinase K (MilliporeSigma) and 7M urea (MilliporeSigma). The RNA-adaptor products were purified by acid phenol:chloroform: isoamyl alcohol extraction and ethanol precipitation. Purified RNAs were then subjected to reverse transcription using Superscript III (Thermo Fisher) with adapter-specific RT primers according to the manufacturer's instruction. Resulted single-stranded cDNA products were purified using ExoSAP-IT (Thermo Fisher Scientific), and ligated to 5' linker in 20 μL of mixture: 1.1 μl H2O, 2 μl 10x T4 RNA ligase buffer (NEB), 1 μl of 5'-linker, 9 μl 50% PEG-8000 and 0.5 μl of T4 RNA ligase 1 (10U/μl – NEB M0204) at room temperature overnight. Resulted linker-ligated cDNA products were purified using MyONE Silane beads (Thermo Fisher Scientific), and PCR-amplifed. The resulting libraries were gel-purified and sequenced with the Illumina HiSeq platform.