GSM1481604: WT NO 3; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
10 min after inoculation & treatment, 3 ml of culture was removed and immediately mixed with 6 ml of RNAprotect Bacteria Reagent (Qiagen). Purification was performed using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. Briefly, after 5 min incubation with the RNAprotect reagent, the sample was centrifuged for 10 min at 5000×g, and the supernatant carefully discarded. Cell pellets were stored at −80 °C until all replicates and conditions for that particular experiment were completed, allowing simultaneous purification of all samples. To eliminate potential DNA contamination, an on-column DNase digestion was performed using RNase-free DNase I (Qiagen). Half a microgram of total RNA from each sample was subject to ribosomal RNA depletion using RiboZero Bacteria kit (Illumina, CA) and then converted to strand-specific RNA-seq library using the automated Apollo 324TM NGS Library Prep System and associated kits (Wafergen, CA) according to the manufacturer’s protocol, utilizing different DNA barcodes for each library. The libraries were examined on Bioanalyzer (Agilent, CA) DNA HS chips for size distribution, and quantified by Qubit fluorometer (Invitrogen, CA). The 12 RNA-seq libraries were pooled together at equal amounts and sequenced on Illumina HiSeq 2500 in Rapid mode as one lane of single-end 67 nt reads following the standard protocol.
GEO Accession
GSM1481604