3'CB-seq of Arabidopsis withou snoRNA depletion
Briefly, the nuclei were washed with resuspension buffer (50% glycerol, 25 mM TrisHCl pH 7.5, 0.5 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.4 U/l RNase inhibitor, 1 g/ml pepstatin A, 1 g/ml aprotinin, and 8 mM -mercaptoethanol), immediately followed by washing buffer (25 mM TrisHCl pH 7.5, 300 mM NaCl, 1 M urea, 0.5 mM EDTA, 1 mM DTT, 1% Tween-20, 0.4 U/l RNase inhibitor, 1 g/ml pepstatin A, 1 g/ml aprotinin, and 8 mM -mercaptoethanol). CB RNA was extracted from chromatin pellet using TRIzol reagent, and subjected to genomic DNA removal by TURBO DNase (Life Technologies), rRNA depletion using a riboPOOL kit (siTOOLs Biotech, PanPlant-10 nmol) and polyA RNA removal by oligo (dT) beads (NEB, S1419). Library was prepared using the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific).