GSM6260345: 03idSERSECLIP3; Homo sapiens; RIP-Seq
Extraction was performed using the RBP-eCLIP Kit (#ECK001, Eclipse Bioinnovations, San Diego, CA). 2x10^7 MDA-MB-231 cells were used for each replicate. Cell culture medium was replaced with ice-cold PBS and cross-linking was performed at 254 nM UV with a setting of 400 mJoules/cm^2. After cross-linking, cells were scraped, washed, counted, centrifuged, and snap-frozen. Cells were lysed, sonicated using a Bioruptor Pico (Diagenode, Denville, NJ), and digested with DNase and Ambion RNase-I Cloned (#AM2294, ThermoFisher Scientific). Immunoprecipitation was performed with mouse monoclonal anti-SerRS antibody (#sc-271032, Santa Cruz Biotechnology) bound to goat anti-mouse IgG magnetic beads (#S1430, New England Biolabs). Size-matched input (SMInput) samples were taken before antibody enrichment. Samples were washed, adapters were added, protein-RNA complexes were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes by wet transfer. The membrane section containing the complexes was cut out and digested with Proteinase mix supplied by the manufacturer. Resulting RNAs were purified and reverse transcribed. Resulting cDNA was purified and adapters were ligated on the ends. cDNA was quantified by qRT-PCR, and libraries were amplified by PCR according to manufacturer instructions. Libraries were gel-purified from a 3% low-melting temperature agarose gel, pooled, analyzed by Agilent TapeStation, and sequenced on a NextSeq 2000 (Illumina).