title: Illumina HiSeq 4000 sequencing; LARP iCLIP in MDAMB231 cell lines
design: LARP iCLIP in MDAMB231 cell lines
library_construction_protocol: A total of ~ 40 million cells per biological replicate of GFP and GFP-LARP6 stably expressing MDA-MB-231 cells were irradiated once on ice with 150 mJ/cm2 of UVC (254 nm), using a Hoefer Scientific UV Crosslinker. A total of 4 replicates of GFP and 6 replicates of GFP-LARP6 were irradiated.  Cell pellets were lysed in iCLIP lysis buffer and diluted to a protein concentration of 1mg/ml. RNA is fragmented in lysate with RNase I at 0.4 U/ml. GFP or GFP-LARP6 was immunoprecipitated with GFP (ab290) or GFP-trap magnetic agarose beads (Chromotek). After SDS-PAGE and membrane transfer, the region corresponding to 75â€“200 kDa protein-RNA crosslinked complexes was excised to isolate the associated RNAs.  Library construction followed the iCLIP protocol. Isolated RNAs were reverse transcribed using primers containing an experimental barcode (5nt, XXXXX) and UMI sequence: /5Phos/ WWW XXXXX NNNN AGATCGGAAGAGCGTCGTGAT 1110 /iSp18/ GGATCC /iSp18/ TACTGAACCGC.