GSM2069212: Sma FLAG; Serratia marcescens; RIP-Seq
Strain SLM17 (encoding Hfq-FLAG) and wild type S. marcescens Db10 (negative control) were grown for 5 hours (mid log) in 500 ml LB medium. Cultures were normalised to an OD600 of 2.0 in a final volume of 50 ml (equivalent to 1x1011 cells) and collected by centrifugation. Cell pellets were washed in 700μl of lysis buffer (20mM Tris pH8.0, 1mM MgCl2, 150mM KCl, 1mM DTT), re-isolated and snap-frozen in liquid nitrogen. Thawed pellets were suspended in 800 μl of lysis buffer with 800 μl of quartz beads (diameter 0.1 mm) and cells broken by vortexing (30 s burst followed by 30 s chill on ice) for 10 min, then centrifuged to remove cells and debris. Supernatants were decanted to fresh tubes and 400 μl of Lysis buffer was added to the pellets, which were vortexed for a further 5 min and supernatant again recovered. The combined supernatants for each sample were centrifuged and transferred to fresh tubes to produce the final cleared cell lysates. Anti-FLAG M2 magnetic beads (Sigma, M8823) were prepared as per the manufacturer’s instructions. 50 μl of beads were added to 800 μl of cleared cell lysate, followed by incubation with agitation for four hours at 4oC. A magnetic stand was used to isolate the beads from solution. The supernatant was removed from each sample and beads were washed a further 3 times with 1 ml of TBS and supernatants pooled for each sample.  Protein/RNA complexes were recovered from the beads by re-suspension in 0.5 ml of lysis buffer followed by phenol:chloroform extraction. RNA was concentrated from the aqueous phase by ethanol precipitation, followed by DNase I treatment using the using the RNase free DNase set (Qiagen). Protein was recovered from the phenol phase by acetone precipitation and resuspended in 100 μl of Laemmli buffer. RNA was quantified using the NanoDrop and protein samples were analysed by SDS-PAGE and anti-FLAG immunoblot to confirm success of the Co-IP. cDNA libraries for the Illumina sequencing platform were constructed by vertis Biotechnology AG, Germany, as described previously (Berezikov et al.,2006), without the RNA size-fractionation step prior to cDNA synthesis. Briefly, RNA samples were polyA-tailed using polyA polymerase. Then, the 5'-PPP termini were converted to 5'-P using tobacco acid pyrophosphatase (TAP) to allow for the ligation of the 5’ end RNA adapter. First-strand cDNA was synthesized by an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. In a PCR-based amplification step using a high fidelity DNA polymerase the cDNA concentration was increased to 20-30 ng/µl. A library-specific barcode for multiplex sequencing was part of a 3'-sequencing adapter.