GSM8684683: A.tauschii, Chromatin_Bound(CB) RNA-seq, AL8-78,leaf, replicate1; Aegilops tauschii; OTHER
Nuclei were isolated from 1 mL tissue powder solubilized in 20 mL ice-cold Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextron T40, 20 mM HEPES, pH7.4, 10 mM MgCl2, 0.75% Triton X-100, 0.5 mM EDTA, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF). The mixture was incubated on ice for 5 min, then filtered through 110 µm nylon mesh and a 40 µm cell strainer. The crude nuclei were spun down at 3,500 g for 5 min at 4°C, followed by washes with 150 µL of resuspension buffer (50% Glycerol, 0.5 mM EDTA, 25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF, 0.4 U/µL RNase inhibitor) and 300 µL of wash buffer (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 M urea, 0.5 mM EDTA, 1% Tween 20, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF, 0.4 U/µL RNase inhibitor). Again, the obtained nuclei were washed with another 150 µL of resuspension buffer and 150 µL of wash buffer. CB RNA was extracted from washed nuclei using TRIzol reagent and subjected to genomic DNA removal by TURBOTM DNase (Life Technologies). After purification with RNA Clean & ConcentratorTM-5 kit (Zymo Research, R1013), ~1 µg CB RNA was subjected to rRNA depletion by a riboPOOL kit (siTOOLs Biotech, PanPlant-10 nmol) and polyA RNA removal by oligo (dT) beads (NEB, S1419). PolyA RNA was enriched from total RNA by oligo(dT) beads. Purified CB RNA and PolyA RNA were transformed into cDNA libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765) and sequenced on an Illumina NovaSeq platform. Chromatin-bound(CB) RNA-seq