title: GSM2459397: IgG-CLIP-seq low RNase I scRNA; Homo sapiens; RIP-Seq
library_construction_protocol: 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. Protein-associated RNAs purified by IP or CLIP 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.
attributes.tag: GEO Accession
attributes.value: GSM2459397