GSM7843073: uba2a-3_4; Arabidopsis thaliana; RNA-Seq
Total RNA from seeds was isolated using a phenol:chloroform extraction protocol. After grinding (30-50 mg dry seeds), samples were resuspended in 500 µl of homogenization buffer (100 mM Tris pH 8.5, 5 mM EDTA, 100 mM NaCl, 0.5% SDS, 1% beta-mercaptoethanol). Samples were centrifuged for 5 min at RT at 8000 g. The supernatant was transferred to the new tubes. Then 250 µl of chloroform was added and samples were shaken for 15 min. Next 250 µl of phenol was added and shakening was repeated, followed by centrifugation for 10 min at 14000 g. The upper phase was transferred to new tubes, and an equal volume of phenol:chloroform:isoamyl alcohol was added, shaken and centrifuged as previously. The upper phase was mixed with 10% of the volume of 3 M sodium acetate (pH 5.2) and an equal volume of isopropanol. After 30 min centrifugation at 20000 g at 4°C the pellet was washed with 70% EtOH and resuspended in water. Subsequently, the Ambion TURBO DNA-free kit (Invitrogen) was used to digest DNA in the samples following the protocol provided by the manufacturer. 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).