title: Sample 22
organism: Hordeum vulgare
description: Protocols: Barley crown tissues were sampled and frozen immediately in liquid nitrogen, and stored at -80°C for RNA-seq analysis or immediately cross-linked in 1 % formaldehyde under vacuum infiltration for 30 min. for nuclei extraction. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M, and vacuum infiltration was extended for 5 min. After that the material was stored at -80°C until use. Purified nuclei were isolated at 4°C from barley cross-linked crown tissues according to a modified method of Moreno-Romero et al. (2017). Plants were grown in pots (H-LSR 4.5 L; 21 cm in diameter and 20 cm in height) filled with a mixture of clay soil and peat (3:1, w/w) under controlled conditions with 16-h photoperiod (16 h light / 8 h dark), 60 % humidity and PAR irradiance of 234 µmol m-2 s-1 (Apollo 8 LED Grow Light). Each pot was weighed daily to maintain soil moisture above 70 % of field water capacity. The number of pots was set to provide material for all studies. Eight seeds were sown per pot, and after germination, the number of plants was limited to five. Two temperature variants were used: (i) optimum temperature (O) (control) 16/8°C (day/night) from sowing to the end of tillering, followed by 20/12°C (day/night) to maturity; (ii) elevated temperature (H) 28°C from sowing to the end of tillering, followed by 20/12°C (day/night) as in the control. Freshly sorted nuclei destined for use in ATAC-seq were spinned down at 1500 g, for 10 min., at 4°C. and stored on ice prior to the transposase integration reaction. Tagmentation was performed in 2x Tn5 buffer (Illumina) using 50k nuclei and hyperactive transposase Tn5 (Illumina). Reactions were performed at 37° for 30 min. with gentle shaking at 1000 rpm and then stopped at 55° for 10 min. in one volume of STOP buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0,1 % SDS, 100 mM EDTA, pH 8.0). To obtain nucleosome-free DNA substrate the samples were subjected to overnight incubation with 2,5 µul Proteinase K (20 mg/ml) at 65°C followed by adding 10 % Triton X-100 (final concentration 1%) to quench SDS. DNA fragments were purified using the ZymoBIOMICS MagBead DNA Kit (Zymo Research), eluted in 20 µul of elution buffer, and then amplified using Next High Fidelity PCR Mix (NEB) and specific primers for 5 to 12 total PCR cycles . The amplified ATAC-seq libraries were, quantified by qPCR with the NEBNext Library Quantification Kit (NEB), purified using AMPure XP beads (Beckman Coulter) and analyzed regarding library concentration with a Qubit and size distribution using TapeStation (Agilent).