GSM1480973: MEF_WT1; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol followed by purification with phenol:chloroform extraction. RNA quality was analyzed using Agilent 2100 bioanalyzer For HITS-CLIP, Quadriceps muscles from WT FVB mice (3 months, n = 3 each genotype) were dissected, frozen in liquid nitrogen, and crosslinked with UV-light (Stratalinker 1800, Stratagene). For MEFs, 10 cm plates (n = 2 each genotype), were UV-crosslinked followed by scraping cells in ice-cold PBS and centrifugation. For immunoprecipitation, protein lysates were denatured by adding SDS (1% final concentration), heating (100°C/5 min) and subsequent dilution to a final SDS concentration of 0.1% in PXL lysis buffer (Chi et al., 2009) prior to the addition of anti-Mbnl1 (rpAb A2764), Mbnl2 (mAb 3B4) and Mbnl3 (rpAb Mb3/7G) antibodies (5 µg each) For PolyA-seq, Reverse transcription was carried out using an oligodT containing reverse primer. DNA libraries of 200-500 bp were obtained and sequenced (Illumina) using an oligo dT-containing sequencing primer for 35 bp reads RNA tags were generated as described (Charizanis et al., 2012) using RNase A (concentrations of 55 U/ml and 0.55 U/ml for high and low RNase, respectively) and cDNA libraries were generated using RNA linkers and primers as described
GEO Accession
GSM1480973