GSM2163871: Sample_MJ358_1_mM_Choline_12_6; Burkholderia thailandensis E264; RNA-Seq
Cells from 1.5 mL of culture were collected by centrifugation and the resultant pellets were resuspended in RNA Protect Bacteria Reagent (Qiagen) and incubated at room temperature for 10 minutes. Cells were collected by centrifugation and resuspended in 50 µL 10 mM Tris, 1 mM EDTA (TE) buffer with 3 mg/mL lysozyme and incubated at room temperature for 5 minutes. To each of these resuspensions, 1 µL 20 mg/mL proteinase K, 0.5 µL of 10% SDS, 1 µL of DNase I (RNase-free, Ambion), and 2 µL of 50 mM MgCl2 were added and samples were incubated for 5 minutes at room temperature.  RNA was then prepared using the Qiagen RNeasy kit according to the manufacturer’s instructions. Eluate from this purification was further treated with 11 µL 10x DNase I buffer and 2 µL DNase I (RNase-free, Ambion) and incubated at 37 oC for 20 minutes to remove contaminating DNA and re-purified using the Qiagen RNeasy protocol with the optional on-column DNase I step. The resulting purified RNA was assessed for purity by PCR and quantified by bioanalyzer chip, depleted of 16S and 23S rRNA by using MICROBExpress™ Bacterial mRNA Enrichment (Ambion), and subsequently reassessed by Bioanalyzer (Agilent Technologies). Illumina TruSeq Stranded Total RNA Library Prep Kit