GSM5049146: iCLIP-seq of GFP-NUDT21, replicate E; Homo sapiens; RIP-Seq
Cells were crosslinked (0.4 J/cm2) at 254 nm with a Stratalinker 1800 prior to immunoprecipitation.  Lysates generated from the crosslinked cells were treated with Turbo DNAse (Ambion) and diluted RNAse I (Ambion) for 5 minutes at 37°C to digest the genomic DNA and trim the RNA to short fragments of an optimal size range. RNA-protein complexes were immunoprecipitated using 100 μL of protein A/G Dynabeads (Life Technologies) and 5-10 μg of indicated antibodies. Following stringent high salt washes, the immunoprecipitated RNA was 3´ end dephosphorylated and a pre-adenylated adaptor was ligated at the 3´ end as described for a previously described enhanced CLIP ligation method (Van Nostrand et al., Nat. Methods 2016) for adaptor ligation. After additional washes, the immunoprecipitated RNA was 5´ end-labeled with radioactive 32P isotopes using T4 polynucleotide kinase (New England Biolabs). The immunoprecipitated complexes were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Protran). For the input sample, the membrane was cut to match the size of the immunoprecipitated material. RNA was recovered by digesting proteins using proteinase K (Thermo Fisher). RNA was reverse transcribed into cDNA using reverse transcription primers that include barcode sequences to enable multiplexing,  a unique molecular identifier, and a BamHI restriction site. The cDNA was size selected (low: 70 to 85 nt, middle: 85 to 110 nt, and high: 110 to 180 nt), circularized to add the adaptor to the 5′ end, digested at the internal BamHI site, and then PCR amplified using AccuPrime SuperMix I (Thermo Fisher). The final PCR libraries were purified on PCR purification columns (QIAGEN).  Eluted DNA was mixed at a ratio of 1:5:5 from the low, middle, and high fractions and submitted for sequencing.