GSM6384112: Col0_2; Arabidopsis thaliana; RNA-Seq
Total RNA was isolated using E.Z.N.A. Plant RNA Kit (OMEGA biotek) following manufacturer instructions. 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. cDNA pool was purified with Ampure beads (1.5x beads to sample volumes) and then 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. Reaction was 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). Reaction was 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, sample was 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 reaction with Q5 2× PCR Master Mix (NEB) and 0.5 uM final concentration of primers. Library was Illumina Nova-seq sequenced to obtain two reads: R1 (contain barcode and UMI) and R2 (contain mRNA sequence).