GSM2970400: WT_NTR_37; Escherichia coli; RNA-Seq
Total RNA was extracted from approximately 5 × 108 E. coli cells using the RNeasy Mini kit (Qiagen, Cat. No. 74104). Briefly, bacterial cell cultures were directly mixed with twice the volume of RNA-protect Bacteria Reagent (Qiagen, Cat. No. 76506) and the recommended protocol of lysozyme-mediated lysis and digestion with Proteinase K was followed. The RNase-Free DNase Set (Qiagen, Cat. No. 79254) was used for an on-column DNase digestion for 30 min prior to RNA elution. The TruSeq RNA Sample Prep kit v2 (Illumina, Cat. No. RS-122-2001) was used.  Total RNA samples (1000 ng) were ribosomal-RNA-depleted using the Ribo-Zero™ Magnetic kit for bacteria (Epicentre, Cat. No. MRZMB126) and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the barcode index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched by PCR.  The quality and quantity of the enriched libraries were validated using the 2200 TapeStation system (Agilent) and quantitative PCR.  The products were a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10 nM in 10 mM Tris-Cl, pH 8.5 at 25ºC supplemented with 0.1% (v/v) Tween-20.  Samples were pooled to equimolar amounts and sequenced in a single lane. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Cat. No. GD-401-3001) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT.  Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Cat. No.  FC-401-3001).