Instrument: Illumina HiSeq 2000
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cell pellets were resuspended in lysis buffer (50mM HEPES-KOH pH7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitor), sonicated and total protein concentrations were adjusted to 1mg/ml. The total protein of both samples, CsrA+2xFlagTag and negative control, was cleared separately by BSA-blocked Dynabeads protein G (Invitrogen) and subsequently incubated with Dynabeads protein G coupled to Anti-Flag antibodies (Sigma) over night at 4°C on a rotating platform. Samples and negative control were washed twice with Lysis buffer containing 350mM NaCl and 5 times with wash buffer (10mM Tris-HCl pH 7.5, 250 mM NaCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA). Subsequently beads were washed with TE buffer, resuspended in elution buffer (50mM Tris-HCl pH 8.0, 1mM EDTA, 1% SDS) and incubated at 65°C for 30 min. Crosslinking was reversed and DNA and protein were digested to get purified RNA. The RNA was metal-catalyzed heat fragmented to a size around 100-200nt using the RNA fragmentation kit and the RNA of both independent samples was purified and further processed according to the TruSeq stranded mRNA sample preparation guide of Illumina. The two parallel processed samples were ligated with adaptor 6 (positive CsrA+2xFlagTag library) and adaptor 12 (negative control, minus FlagTag), respectively, the quantity was determined using a Qubit 2.0 (Invitrogen) and the quality was checked using a Bioanalyzer. High quality libraries were sequenced using an Illumina HiSeq platform. This experiment was done in 5 replicates.