RNA-Seq dsRNA: microbes mineral matrix sample cold water spring
Four ~2g samples of Crystal Geyser Site 5 material preserved in Life Soil Preservation solution (MoBio) at -20oC were thawed on ice, spun at 2500g 5 min at RT and Lifeguard Soil Preservation solution removed, total RNA and DNA was isolated using PowerSoil RNA and DNA Accessory kit (MoBio) following manufacturers instructions.  RNA recovered from the 4 samples was combined. Concentration and purity of RNA was assessed using a nanodrop (Thermo Scientific). dsRNA was isolated by incubating 8g of total RNA with 10 units RNase 1 (Ambion) and 4 units Turbo DNase 1 (Ambion) in 50l 1X Turbo DNase buffer supplemented with NaCl to final monovalent salt concentration of 0.2M at 37oC for 1 hour followed by immunoprecipitation. 25l Protein A Dynabeads (Invitrogen) were washed and bound by 5g of anti-dsRNA J2 antibody (Scicons English and Scientific Consulting, Hungary) then incubated with the ssRNase/DNase treated total RNA in 1X IP buffer (20mM TrisCl pH 7.5, 150mM NaCl, 0.2mM EDTA, 0.1% Tween20) for 2 hours at 4oC with rotation. The beads were collected using a magnetic stand, the supernatant removed, the beads washed 3X with 1X IP buffer and the dsRNA recovered from the beads in 1X IP buffer using Trizol-LS (Ambion) followed by purification using RNA Clean-Up and Concentration Micro-Elute kit (Norgen Biotek Corp.). The amount of dsRNA recovered was determined by dsRNA immunoblotting analysis. Strand-specific cDNA library was prepared from 12ng of dsRNA using Scriptseq V2 library kit (Epicentre) following manufacturers instructions except dsRNA was denatured in nuclease-free water at 95oC 2 min then placed on ice before the fragmentation step and fragmentation was performed for 8 min.