title: GSM2260032: wildtype 2; Escherichia coli K-12; RNA-Seq
library_construction_protocol: Overnight cultures started from three independent colonies of each of three E. coli K1508-derived strains, containing either (i) pGZ119EH; (ii) pPMR132; (iii) pPMR132-E292K, were used to inoculate 20 mL cultures (altogether 9 cultures). At OD600 =0.2, 1 mM IPTG was added to induce the tac promoter of the expression cassette in the plasmids. One hour post-induction, 10 mL of culture was immediately harvested by centrifugation (10 min, 4000 x g at 4°C). Supernatant was aspirated and the pellet suspended in 1 mL resuspension buffer (20 mM NaOAc pH 5.5, 1 mM EDTA; prewashed with phenol:chloroform). An equal volume of cold acid phenol:chloroform (1:1 mix) was added together with acid-washed glass beads (Sigma-Aldrich) to assist cell disruption by vortexing. The aqueous phase was separated by centrifugation (5 min, 13000 x g, 4°C) and washed again in phenol:chloroform, then with phenol and finally chloroform before precipitating nucleic acids by addition of 1/10 volume of 3 M sodium acetate (pH 5.5 and 2.5 volumes of 95% ethanol (molecular biology grade, Merck), overnight at -80°C. Precipitate was collected by centrifugation (30 min, 13000 RCF, 4°C). Pellets were air-dried and suspended in DEPC-treated de-ionised water (Milli Q) containing 1 U/μL Superase-In RNAse Inhibitor (Ambion). Genomic DNA was removed by DNase digest (30 min at room temperature) using TURBO DNase I (Ambion) and repurified by phenol:chloroform as described above. The quantity and quality of extracted RNA was determined by spectrophotometry. Furthermore, capillary electrophoresis (Bioanalyser 2100, Agilent) was used to determine the ratio of 16S to 23S bands and rule out the skew of RNAs towards shorter lengths as a measure of RNA integrity. The quality score assigned based on the electrophoresis profile is expressed as RNA integrity number (RIN) and determined using the Bioanalyser 2100 software; score between 8 and 10 is considered to be acceptable for transcriptomic analysis. Samples with RIN >8.5), were submitted to Ambry Genetics (Los Angeles, USA) for RNA sequencing. RNA sequencing libraries were prepared by Ambry Genetics (Los Angeles, USA). First, ribosomal RNA was depleted from the total RNA samples using the Ribo-Zero rRNA depletion kit (EpiCentre), then directional 100 bp, paired-end RNA sequencing libraries were prepared using the TruSeq stranded sample preparation kit version 2 (Illumina, CA, USA), allowing differentiation of sense from antisense transcripts. These libraries were then sequenced on two lanes of an Illumina HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM2260032