title: NextSeq 500 paired end sequencing; Transcriptome wide mapping of Hfq mediated RNA-RNA interactions in E. coli (RIL-seq)
design: Transcriptome wide mapping of Hfq mediated RNA-RNA interactions in E. coli (RIL-seq)
library_construction_protocol: E .coli Strains MG1655 and HM34 were grown overnight in LB at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB, and re-grown with shaking at 37 °C to stationary phase (6 hr) To trim the exposed parts of the RNAs, beads were resuspended in 200 μl of lysis buffer supplied with 0.5 unit of RNase A/T1 mix (Thermo Scientific) without RNase inhibitor, and incubated for 3-15 min at 22 °C or 37 °C. RNase digestion buffer was removed and the beads were washed three times with 200 μl lysis buffer supplied with 0.1 U of SUPERase INTM RNase Inhibitor (Life technologies) at 4 oC. 5’OH ends were phosphorylated and 2'P / 3'P ends were dephopsphorylated by incubation with 40 units of T4 PNK (New England Biolabs) in 80 μl of PNK buffer for 2 hr at 22 °C. PNK reaction mixture was removed and the beads were washed three times with 200 μl lysis buffer at 4 oC. Neighbouring RNAs were ligated overnight using 216 units of T4 RNA Ligase 1 (New England Biolabs) in 80 μl ligase buffer. Ligation mixture was removed and the beads were washed three times with 200 μl lysis buffer at 4 oC.  Ligation of 3' barcoded adaptors. First cDNA strand was generated using SuperScript III (Life Technologies) RNA was extracted according to the standard LS-TriReagent protocol (Sigma) RNAtag-Seq protocol (Shishkin et al., 2015)