GSM2424749: iCLIP-seq of Nacc1 in N2A cells, replicate 1; Mus musculus; OTHER
Lysates generated from the crosslinked cells were treated with Turbo DNase (Ambion) and RNase I (1:500; Ambion) for 5 min at 37°C to digest the genomic DNA and trim the RNA to short fragments of an optimal size range. A total of 2% input material was saved to prepare size-matched control libraries. RNA-protein complexes were immunoprecipitated using 100 μl of protein G Dynabeads (Life Technologies) and 10 μg of anti-Nac1 (Abcam). Following stringent high salt washes, the immunoprecipitated RNA was 5' end-labeled using radioactive 32P isotopes. Pre-adenylated adaptors were ligated to the 3' end using the conditions reported by van Nostrand et al. (2016; PMID: 27018577). The immunoprecipitated complexes were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Protran). RNA was recovered by digesting proteins using proteinase K and then reverse transcribed into cDNA. Reverse transcription primers and barcodes are indicated in the sample metadata. The cDNA was size selected (low – 70 to 85 nt, middle – 85 to 110 nt, 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 (Life Technologies). The final PCR libraries were purified on PCR purification columns (Qiagen) to remove residual PCR reagents, and a ratio of 1:5:5 from the low, middle and high fractions were submitted for sequencing.
GEO Accession
GSM2424749