HnRNPM_IP_H1
RNA immunoprecipitation (RIP) assay was performed using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA). In brief, hypoxic cells were lysed, lysates were incubated with hnRNPM antibody (sc-20002; 5ug) or control IgG (SC-2025; 5ug) conjugated with magnetic beads (50μl) for 4 h. The protein-RNA complexes were immunoprecipitated and magnetically separated. The RNAs were isolated by RNeasy mini kit (QIAGEN, Hilden, Germany), and treated with a DNA-free kit (Ambion) to eliminate any DNA contamination. The isolated RNAs were reverse-transcribed to cDNA for RT-PCR in the further experiments.   For the RIP sequencing, the isolated RNAs were directly proceeding to library preparation using the Library Builder™ Whole Transcriptome Core Kit on an automation system (the AB Library Builder™ System, Thermo Fisher Scientific Inc., Waltham, MA). The cDNA libraries with size ranged between 200-300 bp were sequenced with read length of 200 bases using the Ion Proton™ sequencer. The overall raw reads were about 29-34 million from the sequencing of input and hnRNPM-precipitated mRNAs. The raw reads were processed with adaptor removal and followed by mapping to the human genome (hg19, released from UCSC database) using both TopHat2 and Bowtie2 tools. To identify the differentially hnRNPM-precipitated mRNA targets, the mapped profiles from the duplicated experiments were analyzed using the Partek Genomics Suite (version 4.9, http://www.partek.com/).