title: GSM4628081: MBS cell line 4; Mus musculus; RIP-Seq
library_construction_protocol: CLIP protocol from (Cho et al., 2012) was used with the following adaptations  15cm dishes of MEFs (at ~70% confluency) were washed with ice cold DPBS, after aspiration and addition of 12mL ice cold DPBS, cells were irradiated with 300mJ/cm of 254nm UV light (BioRad GS Gene Linker). After irridation, each 15cm dish was scraped and collected into a 15mL falcon tube. Cells were pelleted at 500g for 5 minutes and then the pellets were stored at -80 until use. For Immunoprecipitations, a 1:1 mix of monoclonal Anti-GFP antibodies (Sigma Aldrich / Roche 11814460001) were used with protein G dynabeads (4ug of antibodies with 50uL of beads, Thermo Fisher Scientific). All washes were performed using a magnetic stand. CLIP library construction protocol from (Cho et al., 2012) was used. Briefly, RNAse A was used to cleave the RNA prior to 3' adapter ligation. After purification and gel extraction of crosslinked fragments, 5' end adapter ligation was performed. Generation of cDNA, purification, amplification and size selection of amplfied cDNA libraries was performed as per Cho et al, 2012 and then sequenced on NextSeq500 in high output mode.
attributes.tag: GEO Accession
attributes.value: GSM4628081