GSM7990522: Germinating seed, 6h light, rep1, RNA-seq; Arabidopsis thaliana; RNA-Seq
50 mg of sample (or 25 mg for dry seeds) was flash-frozen in liquid nitrogen and ground to a powder with a mortar and pestle. 1.5 mL of extraction buffer (EB; 100 mM Tris-HCl pH 9.5, 150 mM NaCl, 5 mM EDTA pH 8, 1% sarkosyl) was prepared per sample, and 7.5 µL beta-mercaptoethanol was added to each EB aliquot and briefly vortexed. After addition of the EB to the frozen ground tissue powder, the homogenate was mixed vigorously and left on ice for 10 min, with intermittent vortexing. This was followed by a 10 min max-speed centrifugation at 4 ˚C in a benchtop centrifuge. 1 mL of supernatant (avoiding the starchy upper-most layer) was transferred to a new tube and mixed with 1 mL chloroform. Following a 15 s vortexing step, the mixture was centrifuged as before and 900 µL of the upper phase transferred to a new tube. To this was added 90 µL 3 M NaOAc and 1 mL Tris-buffered phenol-chloroform, followed by vortexing for 15 s and another centrifugation as before. As much clear supernatant as possible was moved to a new tube and gently mixed by inversion with an equal volume isopropanol and incubated at 4 ˚C for 1 h. The precipitated RNA was pelleted by a 20 min max-speed centrifugation at 4 ˚C. The supernatant was discarded and the pellet washed with 800 µL ice-cold 75% ethanol, followed by a 5 min centrifugation at 7,500 x g at 4 ˚C. The supernatant was discarded, further removed with a pipette after pulse-spinning, and left inverted to dry for 2 min. The pellet was dissolved in 100 µL nuclease-free water, to which 10 µL DNaseI buffer (ThermoFisher Scientific), 5 µL DNaseI (ThermoFisher Scientific), and 1 µL RiboLock (ThermoFisher Scientific) was added and left to incubate at 37 ˚C for 30 min. The resulting DNA-free RNA was isolated using a modified RNA purification protocol. Briefly, 240 µL nuclease-free water was added to the DNaseI reaction alongside 40 µL solubilization buffer (200 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8, 5% SDS) and vortexed. Afterwards 40 µL 3 M NaOAc and 800 µL Tris-buffered phenol-chloroform was added and again vortexed for 15 s, followed by a max-speed centrifugation for 5 min at 4 ˚C. The supernatant was transferred to a new tube containing 800 µL chloroform and vortexed for 15 s, followed by another centrifugation as before. The supernatant was again transferred to a new tube, this time to one containing 1 mL ice-cold ethanol, gently mixed by inversion, and left at -80 ˚C overnight. The following day the tube was centrifuged as before for 20 min. The supernatant was discarded and the pellet was washed with 800 µL 75% ethanol, followed by a 5 min centrifugation at 7,500 x g at 4 ˚C. The supernatant was removed, and again removed using a pipette after pulse spinning. After leaving the tubes to dry inverted for 2 min, the pellet was dissolved in 27 µL nuclease-free water (of which 1.5 µL was used for quantification with a NanoDrop (ThermoFisher Scientific)) and stored at -80 ˚C. RNA integrity was assessed by running 1 µg RNA in a denaturing RNA gel and 100 ng RNA with a Bioanalyzer RNA analysis chip (Agilent). Total RNA was rRNA-depleted and library preparation was performed using the TruSeq Stranded Total RNA library preparation kit with Ribo-Zero (Illumina) following the manufacturer's protocol.