GSM4704039: Roots CTL rep 2; Phaseolus vulgaris; miRNA-Seq
RNA was extracted with Trizol following manufacture´s recommendations (Thermo Fisher). RNA quality was evaluated by capillary gel electrophoresis on an Agilent 2100 Bioanalyzer using the AgilentRNA6000 Pico kit. Separation of low and high molecular weight RNAs was performed with polyethylene glycol (PEG) precipitation essentially as described by Lu et al (2007). Low molecular RNAs were further separated into a denaturing 15% (w/v) polyacrylamide gel containing 0.48 % (w/v) of urea. The population of sRNAs ranging from 18 to 35 nucleotides was excised from the gel and eluted with 250 μl of 0.3M NaCl by incubating overnight at 4°C. Eluted RNAs were filtered through 5 µm spin-X filter by centrifuging at 16,000 xg. sRNAs (18-35 nucleotides) were precipitated by adding 3 μg of glycogen as a carrier and 2 volumes of 95% (v/v) ethanol and incubating at -80°C for two hours. Samplers were centrifuged for 30 min at 16,000 xg at 4°C and washed with 200 μl of cold 80% ethanol. The supernatants were discharged, and pellets containing the small RNAs were air dry and resuspended into 10 μl of MiliQ water. sRNA-seq libraries were generated using kit TruSeq Small RNA preparation kit following manufacture's recommendations (Illumina). SuperScript II reverse transcriptase (Thermo Fisher Scientific) was used for cDNA synthesis. DNA fragments were purified using AMPure XP beads (Beckman Coulter Genomics) and their size was verified onan Agilent 2100 Bioanalyzer using the DNA-1000 kit (Agilent).
GEO Accession
GSM4704039