GSM7989997: Col0_2; Arabidopsis thaliana; RNA-Seq
RNA extraction from seeds was performed by using the phenol–chloroform protocol. The frozen seeds were ground to a powder with using an electric drill and then mixed in 600µL of RNA extraction buffer (100 mM Tris pH 8.5, 5 mM EDTA ph 8.0, 100 mM NaCl, 0.5% SDS, 1% beta-mercaptoethanol). Afterward, samples were centrifuged for 5 min at 14,000 g at 4°C. The supernatant was transferred to new eppendorf tubes and 250µl of chloroform was added and samples were shaken at room temperature for 15 min. Then 250µl of phenol was added and samples were shaken for a further 15 min and centrifuged for 10 min at 14,000 g at 4°C. Then, 550µL of the aqueous layer was transferred to new tubes and mixed with 550µL of phenol–chloroform–isoamyl alcohol 25:24:1. Samples were shaken for 10 min at RT and centrifuged for 10 min at 14,000 g at 4°C. Then, 500µL of supernatant after transferring to new tubes was mixed with 10% of the volume of 3 M sodium acetate and 80% of the volume of pure ice-cold isopropanol and incubated for 15 min at RT. After the incubation, samples were centrifuged for 30 min at 14,000 g at 4°C. Finally, the RNA pellet was washed in 1mL of 80% ethanol, dried and resuspended in Milli-Q water. DNase treatment of RNA samples was performed with using TURBO DNA-freeTM Kit (Life Technologies), according to the manufacturer's protocol. 500 ng of RNA was used in reverse transcription with 50 mM different barcoded oligo(dT) primers and SuperScript III. 0.25 of each reaction was pooled and pools were purified with Ampure beads (1.2x beads to sample volumes) and eluted in 17 ul of water. 15 ul of recovered cDNA pool was mixed by pipetting with 15 ul of second-strand synthesis mix (2x NEBNext Second Strand Synthesis (dNTP-free) Reaction Buffer (NEB), 1 U RNaseH (NEB), 1 U E. coli DNA ligase I (NEB), 5 U E. coli DNA polymerase (NEB), 30 uM dNTPs) and incubated overnight at 16°C. Reactions were purified with Ampure beads (1.2x beads to sample volumes) and eluted in 4 ul of water. 3 ul of recovered dsDNA sample was used for tagmentation using 3 ul 5x diluted B-loaded Tn5 enzyme (homemade) and 6 ul freshly prepared 2x buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 50% DMF). Reactions were mixed by pipetting and incubated for 7 min at 55°C followed by 5 min at 80°C. Water was added up to 30 ul, samples were purified with Ampure beads (1.2x beads to sample volumes) and eluted with 11 ul of water. Illumina indexing PCR was performed using 10 ul tagmented DNA in 50 ul reactions with Q5 2× PCR Master Mix (NEB) and 0.5 uM final concentration of primers. Libraries were Illumina Next-seq sequenced to obtain two reads: 21 nt R1 (contain barcode and UMI) and 55 nt R2 (contain mRNA sequence).