GSM8223174: timecourse_drought_day1, snRNAseq; Arabidopsis; RNA-Seq
Seedlings were transferred to vermiculite pots two weeks after germination in trays, 6 seedlings per pot. Vermiculite was saturated with ¾ LS liquid media before the seedling transfer, 2L media per tray. After two days each tray was watered with 2L ultra-pure water, and this continued for two weeks until the drought treatment started. When plants were 30 days old, each pot was weighed and transferred to a dry tray. The experiment started when each pot reached to 30% SWC. Between 12-18 whole rosettes were collected for each time point * condition sample. We used mortar and pestle to grind the frozen tissue. Powdered tissue was then placed in a nuclei extraction buffer [NEB; 500ul 1M TRIS pH=7.4 (Cat# 15567027, Thermo Fisher Scientific, MA), 150ul 1M MgCl2 (Cat# AM9530G, Fisher Scientific, MA), 100ul 1M NaCl (Cat# AM9760G Fisher Scientific, MA), 50ml nuclease-free water (Cat# AM9937 Thermo Fisher Scientific, MA), 25ul 1M spermine (Cat# 85590-5G, MilliporeSigma, MA), 10ul 1M sperimidine (Cat# S2626-5G, MilliporeSigma, MA), 500ul proteinase inhibitor (PI; Cat# P9599-5ML MilliporeSigma, MA), 250ul BSA (Cat# B2518-100MG, MilliporeSigma, MA), 250ul SUPERase-In ()] and incubated for 10 min. After incubation, tissue was filtered through a 40um filter (Cat# 43-57040-51, pluriSelect, Germany). We then centrifuged at 500 rcf for 5 min at 4°C. Supernatant was aspirated out and nuclei were resuspended with NEB + 500ul 10% Triton (Cat# 93443-100ML, MilliporeSigma, MA) and no PI. We incubated for 15 min, filtered through a 40um filter and spun at 500 rcf for 5 min. We washed until the pallet was clear. In 15 mL tubes, we prepared the density gradient using the Density Buffer [DB; 120 mM Tris-Cl pH=8 (Cat# AM9855G, Fisher Scientific, MA), 150 mM KCl (Cat# AM9640G, Fisher Scientific, MA), 30 mM MgCl2, 35mL H20 per 50mL] and filter sterilized it. We mixed 5 volumes of Optiprep (Cat# D1556-250ML, MilliporeSigma, MA) and the buffer volume to create a 50% stock. We also made a dilutant stock [400 mM Sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-Cl pH 8, 28mL H20 per 50mL] that we filter sterilized.  We created gradient tubes by preparing two 15ml tubes. A 45% solution was made by mixing 9ml 50% solution and 1ml dilutant, and a 15% solution by combining 1.5ml 50% stock and 3.5ml dilutant. To create the gradient tubes for each sample we poured ~2ml 45% solution and ~1.5ml 15% into each 15ml tube. We gently poured 2ml of the nuclei solution at the top of the density gradient and then spun the tubes at 1,500 rcf for 5 min with no breaks. After the spin, we pipette off nuclei and place them into a 15 ml tube with ~ 6mL of NEB (with no triton or PI).  After counting the nuclei, the nuclei suspension was loaded onto microfluidic chips (10X Genomics) with HT-v3.1 chemistry to capture ~20,000 cells/sample. Cells were barcoded with a Chromium X Controller (10X Genomics). mRNA was reverse transcribed and Illumina libraries were constructed for sequencing with reagents from a 3' Gene Expression HT-v3.1 kit (10X Genomics) according to the manufacturer's instructions. cDNA and final library quality were assessed using Tape-Station High Sensitivity DNA Chip (Agilent, CA). HT-v3.1 10x Genomics