title: GSM6368940: dog1, NaCl, 2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Frozen seeds were ground and mixed with 0.6 ml of homogenization buffer (100 mM Tris pH 8.5, 5 mM EDTA, 100 mM NaCl, 0.5 % SDS, 1% b-mercaptoethanol). Then, 0.6 ml of chloroform was added, vortexed, and centrifuged for 10 min 14,000g. Supernatant was transfered to new tubes and 0.3 ml of phenol was added and vortexed. Then, 0.3 ml of chloroform was added, vortexed, and centrifuged for 10 min 14,000g at 4°C. 0.5 mL of supernatant was transferred to new tubes, mixed with 0.5 mL acidic phenol-chloroform-isoamyl alcohol, vortexed, and centrifuged for 10 min 14,000g at 4°C. Last step was repeated once more. Then, supernatant was mixed with 0.5 mL of chloroform, vortexed, and centrifuged for 10 min 14,000g at 4°C. Finally, the supernatant was mixed with 10% of the volume of 3M sodium acetate and 80% of the volume of pure isopropanol and incubated for 20 min at -80C. The RNA was pelleted by centrifugation for 30 min 14,000g at 4°C, the pellet was washed with 80% ethanol, dried and resuspended in pure water. DNase treatment of RNA samples was performed following the rigorous treatment from TURBO DNase protocol (ThermoFisher) with the following modifications: after adding DNase buffer to the RNA, the samples were mixed by pippeting up and down 10 times and centrifuged 10,000g for 2 min; and DNase incubation at 37C was 30 min plus 20 min. RNA quality was assessed using gel electrophoresis, NanoDrop, PCR and qPCR, before library construction. 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 (control and NaCl pools) 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 Nova-seq sequenced to obtain two reads: R1 (contain barcode and UMI) and R2 (contain mRNA sequence).