alias: GSM2120780
title: GSM2120780: iCLIP-seq of Flag-Zfp871 in N2A cells, clone 14, replicate 2; Mus musculus; OTHER
library_construction_protocol: Lysates generated from the crosslinked cells were treated with Turbo DNase (Ambion) and RNase I (1:500 for Nacc1 and 1:100 for Zfp871; Ambion) for 5 min 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 G Dynabeads (Life Technologies) and 10 μg of anti-Flag or 10 μg of anti-Nac1 (Abcam). Following stringent high salt washes, the immunoprecipitated RNA was 5' end-labeled using radioactive 32P isotopes followed by on-bead-ligation of pre-adenylated adaptors to the 3' end. 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.
identifiers.namespace: GEO
identifiers.id: GSM2120780