GSM4431933: SMInput; Homo sapiens; RIP-Seq
After lysing cells in iCLIP lysis buffer and limited digestion with RNase I (Ambion), ILF3-RNA complexes were immunoprecipitated (ILF3/NF90 antibody from Bethyl Laboratories, A303-651A), and washes were performed. Following RNA dephosphorylation, a barcoded RNA adapter is added to the 3' end. Then after protein gel electrophoresis and nitrocellulose membrane transfer, RNA is isolated from the region of interest by proteinase K treatment. Reverse transcription is performed, and a DNA adapter is added to the cDNA 3' end. (Van Nostrand et al., Nature Methods 13, 508-514. 2016) eCLIP libraries were constructed following previously published ENOCDE eCLIP standard protocol (Van Nostrand et al., Nature Methods 13, 508-514. 2016)