GSM4455861: wt; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RIP-Seq
CoIP-Ab-beads were extracted with PCI. The aqueous layer was precipitated with ethanol and 1:30 3 M sodium acetate at pH 5.2 at -20 °C over-night. The precipitated RNA was pelleted and washed with 500 µl 75% ethanol. The pellet was dried and solubilized in 15.5 µl water. DNA was degraded by DNase I (Thermo Scientific) as described in the manual in presence of RNase inhibitor for 30 min at 37 °C. The sample was diluted with 100 µl water and the RNA was extracted by PCI. The RNA was solubilized to an OD260 of 1 and analysed in the Bioanalyzer (Agilent) for quality and quantity estimation for RNA-seq. Protein samples were recovered from the organic layer by 10 vol methanol precipitation. RNA library preparation was conducted by the Core Unit SysMed (Kristina Döring, Würzburg). 200 ng eluted RNA from the coIP were denatured at 70 °C for 2 min, cooled down on ice, and 3´-end dephosphorylated by 10 U T4-PNK (M0201S, NEB) in the presence of 20 U RNase inhibitor (M0314L, NEB) for 60 min at 37 °C. 1.6 mM ATP and 10 U PNK were added for 5´-end phosphorylation for 60 min at 37 °C. RNA was purified by silica-based columns (Zymo RNA Clean & Concentrator kit, R1013, Zymo Research) and eluted in 15 µl nuclease-free water (436912C, VWR). RNA was denatured again and pyrophosphates were removed from the 5´-end by 10 U RppH (M0356S, NEB) in 1× NEB buffer 2 (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT) in the presence of 20 U RNase inhibitor for 60 min at 37 °C, and again column purified and eluted in 6 µl. For 3´ adaptor ligation the NEBNext Small RNA Kit (E7560S, NEB) was used: 200 ng RNA were mixed with 1 µl 1:3 diluted 3´ SR adaptors for Illumina, and denatured, 1× 3´ ligation reaction buffer was added and 3 µl ligation enzyme mix in a total volume of 20 µl for 60 min at 25 °C. 1 µl 1:3 diluted SR RT-primer for Illumina were added in a total volume of 25 µl. The annealing was conducted at 75 °C for 5 min, 37 °C at 15 min, 25 °C at 15 min, cooling at 4 °C. 1 µl 5´ SR adaptor for Illumina (freshly solubilized, denatured, and cooled on ice), 1 µl 10× 5´ ligation reaction buffer, and 2.5 µl 5´ ligation enzyme mix were added to the reaction and incubated for 60 min at 25 °C. For reverse transcription, 8 µl first strand synthesis reaction buffer, 1 µl murine RNase inhibitor, and 1 µl ProtoScript II reverse transcriptase were added and incubated for 60 min at 50 °C. PCR amplification of reverse transcripts was conducted by addition of 50 µl LongAmp Taq 2× master mix, 5 µl nuclease free water, 2.5 µl 1:3 diluted SR primer for Illumina, and 2.5 µl 1:3 diluted index primers. The reaction was cycled at 94 °C for 30 s, 14× 94 °C for 15s – 62 °C for 30 s – 70 °C for 90 s, 70 °C for 5 min, and cooled at 4 °C.   DNA was purified by MagSi-NGSPREP Plus (MDKT00010005, Steinbrenner Laborsysteme GmbH) beads with a ratio of 1.8× to reaction volume. All steps were executed in DNA-LoBind tubes (#0030108051, Eppendorf). The final supernatant was analysed by Qubit 2.0 (Thermo Scientific) with the dsDNA HS Assay Kit (Q32854, Thermo Scientific) and the Bioanalyzer 2100 (G2939BA, Agilent) with the DNA 1000 kit (5067-1504) or HS DNA 7500 kit (5067-1506). coIP-seq
GEO Accession
GSM4455861