GSM2830183: delta_pSymB_1; Sinorhizobium meliloti; RNA-Seq
Samples were kept on ice or at 4°C throughout the process except where indicated.  Each cell pellet was resuspended in 480 µL of resuspension solution (10 µL of 14.26 M β-mercaptoethanol per 1 mL of nuclease-free ddH2O) and transferred to a nuclease-free 1.5 mL centrifuge tube.  Cell suspensions were then mixed with 480 µL of room temperature hot phenol solution (per 6 mL: 5 mL of hot phenol buffer [20 mM Tris-HCl pH 7.5, 400 mM NaCl, 40 mM EDTA, 1% SDS, in nuclease-free ddH2O] and 1 mL unbuffered phenol), vigorously vortexed for 30 seconds, and incubated in a water bath at 95°C for 1 minute.  Samples were centrifuged at 16,000 g for 10 minutes at 4°C, and the supernatant was transferred to a fresh 1.5 mL nuclease-free centrifuge tube.  The supernatants were treated with two rounds of unbuffered-phenol:chloroform (1:1) extraction, followed by one round of chloroform extraction.  The nucleic acids were then precipitated by mixing the supernatant with one-tenth sample volume of 3 M sodium acetate and 2x sample volume of isopropanol, mixing, and the mixture was incubated on ice for 30 minutes.  The nucleic acids were pelleted by centrifugation at 16,000 g for 10 minutes, the pellet washed with ice cold 70% ethanol, and air dried.  The pellet was resuspended in 80 µL of nuclease-free ddH2O, mixed with 10 µL DNase I buffer and 10 µL DNase I (20 units; NEB catalog # M0303S), and incubated for 20 minutes at room temperature.  The 100 µL sample was then transferred to a fresh 1.5 mL nuclease-free centrifuge tube, extracted once with unbuffered-phenol:chloroform (1:1) and once with chloroform, and the RNA precipitated as before.  The RNA pellet was resuspended in 100 µL water, the integrity of the RNA confirmed as described below, and the remainder of the sample stored at -80°C until further use. RNA libraries were prepared for sequencing using standard Illumina protocols by the Farncombe Family Digestive Health Research Institute located at McMaster University (Hamilton, Ontario, Canada). Ribosomal RNA was depleted using Ribo-Zero rRNA Removal kits (Illumina).
GEO Accession
GSM2830183