The pan-epigenome of barley: ATAC-Seq
Plants were grown in 35-well trays placed in growth chambers under a 16/8-hour light cycle at 20°C day/16°C night temperature for two weeks. Leaf samples were collected 4 hours after lights-on from three individual plants (biological replicates). For developing inflorescence, plants were grown in a greenhouse under the same light and temperature conditions. Developing inflorescences at Waddington scale scores 6.0-7.0, approximately 1.5 cm length (excluding awns), were collected 4 hours after lights-on from two individual plants. Nuclei were isolated by fine chopping the fresh tissues in isolation buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Trion X-100, 5 mM ß-mercaptoethanol) supplemented with 1x Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). Following isolation, nuclei were quantified via flow cytometry, and 75,000 nuclei were subjected to tagmentation using the Tagment DNA Enzyme (TDE1, Illumina) at 37°C for 30 min. Transposed DNA was purified using the MinElute PCR Purification Kit (Qiagen), libraries were amplified with NEBNext® High-Fidelity 2x PCR Master Mix (New England Biolabs), and the final library purification was performed using VAHTSTM DNA Clean Beads (Vazyme). Libraries were sequenced (paired-end, 2 x 151 cycles) on Illumina NovaSeq6000 and Illumina NovaSeq X Plus platforms at IPK-Gatersleben, following manufacturer's protocols (Illumina).
Plants were grown in 35-well trays placed in growth chambers under a 16/8-hour light cycle at 20°C day/16°C night temperature for two weeks. Leaf samples were collected 4 hours after lights-on from three individual plants (biological replicates). For developing inflorescence, plants were grown in a greenhouse under the same light and temperature conditions. Developing inflorescences at Waddington scale scores 6.0-7.0, approximately 1.5 cm length (excluding awns), were collected 4 hours after lights-on from two individual plants. Nuclei were isolated by fine chopping the fresh tissues in isolation buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Trion X-100, 5 mM ß-mercaptoethanol) supplemented with 1x Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). Following isolation, nuclei were quantified via flow cytometry, and 75,000 nuclei were subjected to tagmentation using the Tagment DNA Enzyme (TDE1, Illumina) at 37°C for 30 min. Transposed DNA was purified using the MinElute PCR Purification Kit (Qiagen), libraries were amplified with NEBNext® High-Fidelity 2x PCR Master Mix (New England Biolabs), and the final library purification was performed using VAHTSTM DNA Clean Beads (Vazyme). Libraries were sequenced (paired-end, 2 x 151 cycles) on Illumina NovaSeq6000 and Illumina NovaSeq X Plus platforms at IPK-Gatersleben, following manufacturer's protocols (Illumina).