GSM2756428: eCLIP_invitro_B1_noxlink; synthetic RNA; RIP-Seq
Cloning and purification of recombinant hnRNP B1 was performed as previously described (Meredith et al. 2016). HOTAIR and antisense luciferase control RNA (see (Meredith et al. 2016) for sequence details) were in vitro transcribed using the MEGAscript T7 Transcription Kit, treated with TURBO DNase, and purified with RNeasy Qiagen Kit. In a 1:10 RNA:protein molar ratio, 1.2 ug of control or HOTAIR RNA was incubated with recombinant B1 in 100 µL RNA refolding buffer (20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.2 EDTA pH 8.0. 20% Glycerol, 0.5 mM PMSF, 0.5 DTT) for 20 minutes at room temperature. The mixture was diluted to 250 µL in refolding buffer and UV-crosslinked twice in one well of a 24-well plate at 250 mJ and 254 nm wavelength, with mixing by pipette in between. B1-RNA crosslinked and non-crosslinked samples were treated with 0.1 ng RNase A for 3 minutes at 37ºC and 1200 rpm, then stopped with 200 U Murine RNase Inhibitor (NEB). Protein-RNA complexes on nitrocellulose were then digested with Proteinase K (Roche) and reverse transcribed using SuperScript IV (Thermo Fisher). These cDNAs were then prepared for sequencing as described using Illumina TruSeq HT dual indexed primers, and sequenced on an Illumina NextSeq 500 for high-output 2x75 bp run.
GEO Accession
GSM2756428