GSM9251059: Wildtype Col-0 Arabidopsis roots, hypoxic, rep 1; Arabidopsis thaliana; RNA-Seq
Nuclei were isolated from fresh root tissue (1 cm root tips) using a modified protocol based on Guillotin et al. (Guillotin et al., 2023). Roots were collected from 5 different plates and immediately transferred into a pre-chilled Petri dish containing 300 µL of lysis buffer [0.3 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 15 mM Tris-HCl, 20 mM MES, 10 mM MgCl₂, 60 mM KCl, 15 mM NaCl, 0.5 mM spermine and spermidine, 0.05% Triton X-100, 5 mM DTT, 1 mM PMSF, 1% Protease Inhibitor Cocktail (Sigma P9599), 0.4 u/µl Protector RNase Inhibitor (Sigma), 0.4% BSA, 1% DEPC] and chopped on ice with two razor blades for 2–3 minutes. The homogenate was transferred to a chilled microcentrifuge tube, gently ground with a plastic micropestle, and incubated on ice for 2–3 minutes. The suspension was filtered through a 20 µm strainer into a chilled 1.5 ml Eppendorf tube, rinsed with 200 µL lysis buffer, and centrifuged at 1000 × g for 10 minutes. The supernatant was gently removed, and the pellet resuspended in 300 µL of wash buffer [0.3 M sucrose, 15 mM Tris-HCl, 15 mM MES, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM spermine and spermidine, 0.05% Triton X-100, 5 mM DTT, 1 mM PMSF, 1% Protease Inhibitor Cocktail, 0.2 u/µl Protector RNase Inhibitor, 0.4% BSA] and centrifuged at 1000 × g, 5 minutes. The pelleted nuclei were resuspended in 50–100 µL of final buffer [0.3 M sucrose, 15 mM Tris-HCl, 15 mM MES, 60 mM KCl, 15 mM NaCl, 0.5 mM spermine and spermidine, 5 mM DTT, 1% Protease Inhibitor Cocktail, 0.2 u/µl Protector RNase Inhibitor, 1% BSA]. The suspension was filtered through a 10 µm strainer into a fresh chilled tube. Single-nuclei RNA-sequencing libraries were prepared using the 10X Genomics (Pleasanton, CA, USA) Chromium Next GEM Single Cell 3' Reagents Kits v3.1 (Dual Index) protocol. A total of 902 million 100 bp paired-end reads were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA, USA) at Génome Québec (Montréal, QC, Canada).