GSM9288115: ΔlsrL mutant cells, replicate 2 (RNA-seq); Streptomyces venezuelae; RNA-Seq
Using the cell pellets that were set aside from the cultures used for IPOD-HR and RNApol ChIP-seq, RNA was extracted from three to five replicates each of WT, Δlsr2, ΔlsrL, and Δlsr2/ΔlsrL mutant strains. RNA was isolated as described in Moody et al. (2013), using a modified guanidium thiocyanate protocol (Chomczynski and Sacchi, 1987). Primers amplifying a 144 bp region of rpoB (vnz_21470) were used for PCR checks, alongside a quantified chromosomal DNA control, to confirm the absence of DNA contamination. RNA quality was assessed first by agarose gel electrophoresis, and subsequently using an Agilent 2100 Bioanalyzer. In the case where RNA was successfully extracted from more than three replicates, those with the highest quality were used for sequencing. Purified RNA was depleted of rRNA using the Bacterial NEBNext rRNA Depletion Kit (New England Biolabs). Library preparation was conducted using the NEBNext Ultra II Directional RNA kit (New England Biolabs). The samples were pooled into libraries and were sequenced on an Illumina NextSeq 2000, P3, in a 2×50bp configuration at the McMaster Genomics Facility.