GSM5176037: NHDF hnRNPC iCLIP (repl.2); Homo sapiens; RNA-Seq
hnRNPC-RNAs complex was resolved on a gradient NuPAGE gel and blotted onto nitrocellulose membrane. Post-autoradiography the membrane was cut between 48-100kDa size and Proteinase K treatment was performed to isolate RNA.  The reactions were stopped with 1V of Urea buffer and incubated for additional 20 min. RNA was purified by adding equal volumes of Phenol chloroform isoamylalcohol and precipitated with 3V of 100 % ethanol overnight at -20°C. RNA pellets were washed 2x with ice cold 80% ethanol and resuspended in 8μl of RT annealing mixture. The samples were denatured three min at 70°C and cooled to 25°C for 10 min. 12 μl RT reaction mix was added and reverse transcription reaction was carried out. The samples were snap-chilled on ice and passed through the QIAquick columns (QIAGEN) following the manufacturer's protocol. DNA was eluted in 20μl ddH2O. DNA circularization was carried in a final reaction volume of 40 μl of the circligaseII reaction mix at 60°C for 1hour. The circularised samples were supplemented with oligo annealing mix and incubated 4 min at 95°C. Post hybridization the reaction mix was supplemented with 3μl of BamHI and incubated for 1hour at 37°C. Linearized cDNA was further purified using the QIAquick columns. cDNA PCR amplification was carried out and the PCR products were resolved by a gel electrophoresis. The fragments of 180-300 bp were excised and purified. The purified library was analyzed by 75bp single end high-throughput sequencing at Illumina HiSeq2500