GSM5194404: HEK293 cells_cytosol_IgG_rep3; Homo sapiens; RIP-Seq
RNA immunoprecipitation to examine RNAs bound to RNA binding protein was performed according to the nature protocol. Briefly, HEK293 cells were harvested in polysome lysis buffer (100mM KCl, 5mM MgCl2, 10mM HEPES, pH7.0, 0.5% Nonidet P-40, 1mM DTT, 100Uml-1 RNasin RNase inhibitor, 2mM vanadyl ribonucleoside complexes solution), protease inhibitor cocktail. Cell lysates were incubated on ice for 10 min, then centrifuged at 16,000g for 15min at 4C. The supernatants were incubated with dynabeads G that were already incubated with antibodies targeting hnRNPA1 or normal IgG controls (Invitrogen) for 4h at 4C with rotation. Beads were washed with polysome lysis buffer and polysome lysis buffer including 1M urea four times, respectively. The washed beads were incubated with polysome lysis buffer with 0.1% SDS and 30mg proteinase K at 50C for 39min. The bounds RNAs were eluted and subjected to reverse transcription followed by RT-PCR or next generation sequencing to identify individual mRNAs isolated with the hnRNPA1. Total RNA from RNA immunoprecipitation was checked for degradation in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Libraries were prepared using a TruSeq Stranded Total RNA kit from Illumina (Illumina, San Diego, CA, USA); given the high level of degradation, no further fragmentation was necessary during library preparation, and 12 cycles of final enrichment PCR were run for all samples regardless of the starting amount of RNA used. Final library products were quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the fragment size distribution was determined with the Bioanalyzer 2100. All but one of the samples yielded good libraries, though final molar concentrations varied between them. The libraries were pooled equimolarly, and the final pool was quantified via qPCR using the NEBNext Library Quant Kit for Illumina (New England BioLabs, Ipswich MA), according to manufacturer's instructions. The pool was sequenced in an Illumina NextSeq 550 platform, in Paired-End 2x150 cycles format. Sample QC, library preparation and sequencing were performed at the USC Genome Core.