GSM1517846: DGCR8-CLIP-seq replicate 4 (Low RNase 1 treated); Homo sapiens; RIP-Seq
Two million cells were resuspended in 250 μl lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing EDTA-free protease inhibitor cocktail (Roche) and RNase inhibitor (Ambion) and briefly sonicated by using the Bioruptor. The lysates were treated with 5 μl of Turbo DNase (Ambion) and 10 μl of 10 U/μl or 1 U/μl RNase I (Ambion) at 37°C for 3 min. Cellular debris was removed by centrifugation at 13,000 rpm for 15 min. Protein-RNA complexes were immunopurified with an anti-Flag or anti-DGCR8 antibody (Bethyl laboratories) bound to Protein A/G Dynabeads. Beads were washed twice with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) followed by two washes with PNK buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, and 0.2% Tween-20). For dephosphorylation of 3’ ends, 1U of FastAP alkaline phosphatase (Fermentas) was added to the beads and incubated at 37°C for 20 min. After washing the beads twice with PNK buffer, protein-bound RNAs were ligated to a pre-adenylylated 3’ adaptor overnight at 16°C. Beads were washed twice with PNK buffer again and labeled with [γ- 32P] ATP for visualization. The labeled RNAs were separated by 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane, and excised from the membrane. The protein was degraded by proteinase K treatment, and RNA was recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. H1 cells were washed twice with PBS and cross-linked with 1% formaldehyde at room temperature for 10 min. After washing twice with PBS, cells were collected and sonicated in 0.2 ml lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 0.5% Empigen BB). After centrifugation at 13,000 rpm for 15 min, the supernatant was diluted 2.5-fold in IP buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) and incubated with an anti-DGCR8 antibody or IgG bound to Protein A/G Dynabeads at 4°C for 2 hours. Beads were serially washed with 1 ml washing buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), washing buffer II (20 mM Tris-HCl pH 8.0, 2 mM EDTA), washing buffer III (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 1% deoxycholate, 0.25 M LiCl), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were then treated with 5 μl of Turbo DNase at 37°C for 3 min and washed twice with PNK buffer. FastAP alkaline phosphatase was added and incubated at 37°C for 20 min. Beads were washed twice with PNK buffer again. DGCR8-bound RNAs were ligated to a pre-adenylylated 3’ adaptor overnight at 16°C and washed twice with PNK buffer. Protein-RNA complexes were removed from the beads through 30 min incubation with 1% SDS. Crosslinked proteins were eliminated by proteinase K treatment at 65°C for 2 hrs, and RNA was recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. For AGO2 immunoprecipitation flag-AGO2 H1 cells were lysed in IP buffer containing 1% Empigen BB, 0.1 mM DTT, and protease inhibitor cocktail (Roche) in PBS. After centrifugation at 13,000 rpm for 15 min, the supernatant was incubated with an anti-Flag antibody (Wako) or IgG bound to Protein A/G Dynabeads (Invitrogen). Beads were washed five times with IP buffer. AGO2-bound RNAs were extracted with phenol/chloroform/isoamyl alcohol (Sigma) and precipitated with ethanol. Protein-associated RNAs purified by IP, CLIP, or RIP were ligated to a pre-adenylylated 3’ adaptor that contains a 2-nt adapter code for de-multiplexing and a 3-nt random sequence to minimize the loss of information after collapsing identical sequences.  The RNA-adapter hybrids were converted into cDNAs with RT primer and SuperScript III reverse transcriptase (Invitrogen). The cDNAs were size-fractionated by electrophoresis on a 12% denaturing polyacrylamide gel and circularized by single-stranded DNA ligase (Epicentre). Circularized cDNAs were amplified with PCR primers, in which one of them contained a 6-nt primer code for de-multiplexing. PCR products were purified by agarose gel elution and subjected to next-generation sequencing.
GEO
GSM1517846
GEO Accession
GSM1517846