GSM6753634: III_C4_S28; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
Bacterial RNA was isolated from Salmonella strain SL1344. RNA extraction was performed with 1.8 ml of each in vitro culture using the TRIzol reagent (Invitrogen) according to the manufacturer's recommendation. RNA quality was checked using Qubit RNA High Sensitivity Assay Kit (Invitrogen) and 2100 Bioanalyzer RNA 6000 Pico/ Nano kit (Agilent Technologies). Prior library preparation, DNAse treatment was performed using DNAse I kit (Thermo Fisher) followed by ribosomal RNA depletion. Ribosomal RNA (rRNA) was depleted using Lexogen's RiboCop META rRNA Depletion Kit protocol according to manufacturer's recommendation using 100ng total RNA as input per sample. cDNA obtained from single-cells after MATQseq was further processed for library preparation including ribosomal depletion protocol. Library preparation was done using the Nextera XT DNA Library Preparation Kit (Illumina) including DASH protocol (according to (Prezza et al. 2020) with several modifications). Tagmentation was performed with only ¼ of reaction and 0.5 ng cDNA input according to manufacturer's recommendations. Index PCR was performed with 13 cycles using IDT for Illumina Nextera DNA Unique Dual Indexes (Illumina). Obtained libraries were purified with AMPure XP beads (Beckman Coulter) at a 1:1 v/v ratio to ensure capturing sRNA-derived cDNA. After QC, up to 12 samples were pooled equimolar for ribosomal depletion. sgRNA/Cas9 complex formation is followed by DASH using the appropriate ratio of cDNA : Cas9 : sgRNA. Cas9 enzyme was inactivated by Proteinase K (15 min at 37 °C). Afterwards, Proteinase K was inactivated by adding PMSF (1 mM final concentration). Depleted cDNA was purified by another round of AMPure XP bead clean-up and used as input for second PCR amplification. 0.5 ng depleted cDNA was used as input for Nextera XT reactions omitting the tagmentation steps. As primers i5 and i7 index-independent primers were used to amplify non-cleaved cDNA products. PCR was done with 13 cycles and clean-up was performed with AMPure XP beads at a 1:1 v/v ratio