title: GSM1428954: control from OD 0.5 (exponential) conditions; Agrobacterium tumefaciens; RIP-Seq
library_construction_protocol: Co-immunoprecipitation (coIP) experiments of Hfq3xFlag and bound RNAs were based on the procedure described in (Pfeiffer et al., 2007) with minor changes. 100 ml of A. tumefaciens wild-type and hfq3xFlag cultures grown to OD600nm 0.5 and 1.0 in YEB medium or under non-induced (+DMSO) and virulence-induced (+acetosyringone) conditions in AB medium were harvested and resuspended in 2 ml ice-cold lysis buffer (20 mM Tris (pH 7.5), 150 mM KCl, 1 mM MgCl2, 1 mM DTT). Cells were disrupted by French® Press (3 passes, 16,000 psi) and centrifuged at 10,000 x g, 4 °C, for 1 h. 10 ng monoclonal ANTI-3XFLAG® M2 antibody (Sigma-Aldrich, Germany) were coupled to 50 µl Dynabeads® Protein G (ThermoFisher Scientific, Life TechnologiesTM, USA) as described in the instruction manual, and incubated with the supernatant (3 h, 4 °C). Dynabeads® were separated on a magnet and washed 5x with PBS buffer. RNA was isolated using phenol/chloroform/isoamylalcohol and chloroform/isoamylalcohol followed by precipitation with ethanol and sodium acetate. After precipitation remaining DNA was digested by DNaseI (ThermoFisher Scientific, Life TechnologiesTM, USA) and RNA was precipitated as described before. Preparation of cDNA libraries was performed at Vertis Biotechnology AG (Germany). Equal amounts of RNA samples were poly(A)-tailed and 5’-PPP were removed. The RNA adapter was ligated to the RNA 5’-monophosphate and reverse transcription was performed with oligo(dT)-adapter primers resulting in first-strand cDNA. Higher yields of DNA (20-30 ng µl-1) were gained by further PCR-based amplification using primers designed for TruSeq according to recommendations for Illumina (HiSeq). For multiplex sequencing a library specific barcode was part of the 3’- sequencing adapter. Purification was achieved using the Agencourt AMPure XP kit (Beckman Coulter Genomics, USA), followed by capillary electrophoresis.