GSM3674979: BTX642 Leaves, 42 days old, Preflowering drought stress, plot 6; Sorghum bicolor; RNA-Seq
Frozen root and leaf tissue were ground in a cryogenic Freezer Mill (SPEX SamplePrep 6875D, Metuchen NJ USA) for 2-3 cycles of 2-3 minutes, with 1 minute cooling in between. Samples were then stored at -80C. RNA was extracted using the QIAGEN miRNeasy mini kit (Cat. #AM217004) with modifications: 1 mL QIAzol (Cat) and 100 uL 10% Sarkosyl solution were added to 100 mg of frozen tissue and placed in a vortex shaker adapter for 5 minutes. To these, 200 uL chloroform was added, and the samples were then vortexed and incubated for 3 minutes at room temperature before centrifugation at 12,000 xg for 15 min at 4C. The aqueous phase was then transferred to a fresh tube, and ethanol was added to a final concentration of 60%. The remaining procedure was performed according to the manufacturer's recommendations. DNA contamination was removed from each sample using the TURBO DNA-free kit, (Cat. # AM1907, Invitrogen) according to the manufacturer's recommendations. Stranded cDNA libraries were generated using the Illumina Truseq Stranded RNA LT kit. mRNA was purified from 1 ug of total RNA using magnetic beads containing poly-T oligos. mRNA was fragmented and reversed transcribed using random hexamers and SSII (Invitrogen) followed by second strand synthesis. The fragmented cDNA was treated with end-pair, A-tailing, adapter ligation, and 8 cycles of PCR. qPCR was used to determine the concentration of the libraries.
GEO Accession
GSM3674979