title: GSM6166332: SD_L_3d_02; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: For single seed analysis, 16 seeds were sparsely placed on a cooled glass microscopic slide and crashed using another glass microscopic slide. 2 ul of cold water was fast dropped on each seed debris on both slides. Matching two parts of each seed debris were mixed with water by pipetting and combined. 3 ul of seed extract was transferred to the PCR tube on ice. After all seeds were processed, 2 ul of digestion buffer (10 mM Tris-HCl pH 8.0, 1% Triton X-100, 1% Tween 20, 0.5 mM EDTA, 1.3 mg/ml proteinase K) and 1 ul of 10 mM barcoded oligo(dT) primer were added and mixed. Samples were incubated for 10 min at 65°C and 1 min at 85°C and placed on ice. For isolation of RNA from seed pools, frozen seeds were ground and mixed with 1 ml of homogenization buffer (100 mM Tris pH 9.5, 5 mM EDTA, 150 mM NaCl, 1 % sarkosyl, 0.5% b-mercaptoethanol). Seeds debris was centrifuged for 5 min 11,000g, the supernatant was transferred to fresh probe with 500 ul of chloroform and vortexed for 2 min. Then 500 ul of acid phenol was added and again vortexed for 2 min followed by centrifugation for 15 min 11,000g at 4°C. 900 ul of upper phase was transferred to a new tube and mixed with 90 ul of 3M sodium acetate and 600 ul of isopropanol. After 10 min incubation, RNA was pelleted by centrifugation for 15 min 11,000g at 4°C, the pellet was washed with 80% ethanol, dried and resuspended in 1ml of TRIzol. From that point, the procedure follows the standard protocol for RNA isolation with TRIzol. For single seed libraries, digested extracts were mixed with 4 ul of reverse transcription mix (2 mM dNTPs, 2x SuperScriptIII reaction buffer, 10 mM DTT, 50 U of SuperScriptIII, 10 U of RiboLock), incubated for 30 min 42°C, 15 min 70°C and placed on ice. 32 cDNAs obtained using different barcoded oligo(dT) primers were pooled together, 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), 3 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 21 nt R1 (contain barcode and UMI) and 55 nt R2 (contain mRNA sequence). For transcriptomic analysis of seed pools, isolated RNA was treated with Turbo DNase (ThermoFisher) and 500 ng of it was used in reverse transcription using 50 mM different barcoded oligo(dT) primers and SuperScript III. 1/4 of each reaction was pooled and single library was prepared and sequenced exactly as above.