GSM9420788: E. coli Δrnb Δrnr Δfbp, 2 min, rep2; Escherichia coli; RNA-Seq
Following rifampicin treatment, RNA was isolated as previously described [S. Lin-Chao, S. N. Cohen, The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. Cell 65, 1233–1242 (1991)] RNA quality was determined using an Agilent 5200 Fragment Analyzer and only samples with an RNA Quality Number of 8.0 or higher were used for subsequent steps. Before rRNA depletion, ERCC ExFold RNA Spike-In Mix (Thermo Fisher Scientific, Cat. No. 4456739) was added to each sample. In brief, 2 µg of total RNA was combined with 4 µL of a 1:100 dilution of the ERCC RNA spike-in in a final volume of 50 µL. Ribosomal RNA was depleted using the RiboMinus Bacteria 2.0 Transcriptome Isolation Kit (Thermo Fisher Scientific, Cat. No. A47335) following manufacturer's instructions. Next, cDNA synthesis and library preparation were performed using KAPA mRNA HyperPrep Kit (Roche, Cat. No. KK8581) following the manufacturer's protocol. Briefly, rRNA-depleted samples were fragmented into 200–300 bp fragments, and cDNA was synthesized and A-tailed. Then, Illumina TruSeq RNA UD Index V2 dual indexed adapters (IDT, Cat. No. 20042114) were ligated to the A-tailed cDNA and amplified by 12 PCR cycles. Library concentration was measured using a Qubit dsDNA HS Assay Kit (Invitrogen, Cat. No. Q32854) on a Qubit 3.0 Fluorometer (Invitrogen, Cat. No. Q32866). Final library quantification was performed with a qPCR-based method using the KAPA Library Quantification Kit (Roche, Cat. No. KK4824) on a BioRad CFX Opus 96 real-time PCR machine. The strand-specific RNA-sequencing libraries were pooled and sequenced on an Illumina NovaSeq X Plus sequencer with paired-end, 150 cycle reads, with an average of 5 million reads per sample.