3'CB-seq of Arabidopsis with 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). After 3 adapter ligation, the 3 adapter ligated RNA was fragmented to 30-200 nt at 94C for 7 min with 1x first-strand synthesis buffer (Takara), followed by RppH and T4 PNK treatment. After 5adapter ligation, the RNA sample was hybridized with snoRNA probe mix (single strand DNA oligos), and digested with RNase H (Lucigen, H39500).