GSM4104282: 142_NMS1: Mutant (quorum sensing), nitrate, biological replicate 1; Methylobacter tundripaludum 21/22; RNA-Seq
Once samples reached OD600 ~ 0.6-0.7, 2.5 mL of a stop solution of 5% (v/v) phenol in ethanol was added to 22.5 mL of culture. Cells were pelleted for 15 min at 4°C, and resuspended in RNA extraction buffer (a 1:3 ratio of 5% cetrimonium brimide in 2.5 M NaCl to 0.1 M phosphate buffer at pH 5.8). An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1 ratio) was added, and sodium dodecyl sulfate and N-lauroylsarcosine sodium salt were each added at 0.5% (v/v). Cells were lysed in a bead beater with 0.1-mm zirconia-silica beads (Biospec Products, Bartlesville, OK, USA) for 4 min, then centrifuged at 14,000 x g for 5 min. The aqueous fraction was then washed with chloroform-isoamyl alcohol (24:1 ratio), and the aqueous fraction from this wash was precipitated overnight with 150 mM sodium acetate (pH 5.5), 1.5 mM MgCl2, and 50% isopropanol at -80°C. Precipitated RNA was harvested at 14,000 x g for 30 min at 4°C, and treated with DNase I (Ambion, Foster City, CA, USA) before purification with the RNeasy minikit and on-column DNase (Qiagen, Hilden, Germany). cDNA library preparation and RNA sequencing was performed by GENEWIZ using Illumina HiSeq 2x150 (pair ended) reads.