GSM2702786: Nodule_wild_type_3; Pisum sativum; RNA-Seq
Nodules were collected from pea plants 25 days post-inoculation with R. leguminosarum.  Plants were removed from the vermiculite-sand mixture, placed on ice, and nodules were picked from the roots and immediately flash frozen in liquid nitrogen.  Each biological replicate consisted of nodules from three plants from a single Leonard assembly, and enough nodules were collected to fill a 2 mL centrifuge tube to approximately the 500 µL mark.  Three biological replicates were prepared per strain, and nodules were stored at -80°C until use.  For RNA isolation, all nodules per sample were placed in an autoclaved mortar together with 800 µL of 1% β-mercatoethanol in nuclease-free water (Bioshop) and immediately crushed using an autoclaved pestle.  Approximately 500 µL of crushed nodule solution was transferred to a 1.5 mL centrifuge tube on ice, and mixed with 500 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).  The mixtures were vigorously vortexed for 20 seconds then immediately 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.  Each RNA pellet was then resuspended in 100 µL water.  A second round of DNase I treatment, extraction, and RNA precipitation was performed as described above.  The final RNA pellets were each resuspended in 100 µL of water, the integrity of the RNA confirmed with PCR and MOPS-formaldehyde gels (and later with BioAnalyzer), and the remainder of the samples 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).  Depletion of the plant and bacterial rRNA was performed using Illumina ribo-zero kits with a 50:50 mixture of plant and bacteria probes.