GSM8146852: HEK293T, ALAS1-KO, rep2 (Ago2 CLEAR-CLIP); Homo sapiens; RIP-Seq
One 25 cm dish of confluent HEK293T and ALAS1-/- cells 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). Samples were then phosphorylated at 37°C for 20 minutes in 50 μl of PNK mixture: 41.8ul H2O, 5 μl 10X PNK buffer (NEB), 1 μl RNaseOUT, 1.67 μl ATP (30 mM), 0.5 μl T4 PNK - 3' phosphatase minus (NEB M0236L). The immunoprecipitated materials were washed with PNK wash buffer, and then miRNA-mRNA ligation was carried out overnight at 17°C in 100 μl of mixture: 49.25 μl H2O, 30 μl 50% PEG-8000 (NEB #B1004), 10 μl 10x T4 RNA ligation buffer (NEB), 2.5 μl RNaseOUT, 1 μl ATP (100mM), 1 μl BSA (10 mg/ml), 6.25 μl T4 RNA ligase 1 (10U/μl – NEB M0204). The RNAs in the immunoprecipitate were dephosphorylated on the beads with FastAP Thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and ligated to preadenylated adaptors in 40 μL of mixture: 17 μl H2O, 4 μl 10x T4 RNA ligase buffer (NEB), 1 μl of 3'-linker, 16 μl 50% PEG-8000, 1 μl RNaseOUT and 1μl T4 RNA ligase 2 truncated K227Q (NEB M0351) at room temperature for 5 hrs. Samples were then radiolabeled in 50 μl mixture: 5 μl 10X PNK buffer (NEB), 1 μl RNaseOUT, 1.5 μl γ-32P-ATP (15 μCi), 1 μl PNK enzyme (NEB M0201) and 41.5 μl H2O for 5 min at 37°C. 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), followed by circularization using CircLigase (Illumina), and PCR-amplified. The resulting libraries were gel-purified and sequenced with the Illumina HiSeq platform. CLEAR-CLIP