GSM5016585: SRSF2_hpc.WT-2_INPUT; Homo sapiens; OTHER
Total RNA was extracted using Trizol and zymo's direct-zol RNA extration Kit. 10M cells were UV-crosslinked, lysed and sonicated. RNA was fragmented and protein-RNA complexes were immunoprecipitated with a V5 antibody. Inputs (2% of lysate) were saved and run alongside IP samples. IP samples were stringently washed, and for all samples the RNA was dephosphorylated,, followed by on-bead ligation of barcoded RNA adapters to the 3' end.. RNA-protein complexes were run on standard protein gels and transferred to nitrocellulose membranes where the RNA in the region 35 kDa – 110kDa was excised off the membrane and proteinase K treated. RNA was then reverse transcribed followed by treatedment with ExoSAP-IT to remove excess oligonucleotides. Samples were cleaned up with Dynabeads MyOne Silane and subject to qPCR to determine the appropriate number of PCR cycles.