GSM7507178: E. coli + tagged yfif, replicate 3, CL; Escherichia coli; OTHER
Cells were mechanically lysed with glass beads in CLIP lysis buffer. The suspension was cleared once and sonicated on low for 10 cycles (30 s on/off). RNA was partially digested with RNase I prior to immunoprecipitation of the protein of interest. Crosslinked RNA was 3' end dephosphorylated and ligated with a fluorescent adaptor on the beads prior to SDS-PAGE and nitrocellulose transfer. Relevant sections of the membrane were cut out, dissolved and the RNA released by proteinase K treatment. Enriched RNA was reverse transcibed with SSIV (Invitrogen) and treated with RNase H (NEB). cDNA was then column-purified and circularised with circLigase (Epicentre/Illumina) for 2 hrs. Circularised cDNA was directly PCR amplified and quantified with Bioanlayzer prior to sequencing.