GSM8125033: at-endosperm-H3_replicate1_col_Cm_x_ler; Arabidopsis thaliana; OTHER
We coupled the previously published INTACT protocol for endosperm-nuclei purification (Moreno-Romero et al., 2017) with Cleavage Under Targets & Tagmentation (CUT&Tag) (Kaya-Okur et al., 2019). Tissue homogenization and nuclei purification were performed as previously described (Del Toro-De León & Köhler, 2019) except that MgCl2 was replaced by spermidine 0.5 mM in the Honda buffer to prevent pAG-Tn5 activation by residual MgCl2. All steps were performed with streptavidin-bound nuclei. INTACT-purified endosperm nuclei from 250 mg of siliques at 4DAP were resuspended in Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin, 2 mM EDTA pH 7.5). Primary antibody incubation was performed overnight at 4°C with gentle shaking using anti-H3K9me2 (Diagenode Cat. No. C15410060), Anti-Histone H3 (Sigma-Aldrich Cat. No. H9289) or anti-H3K27me3 (Cell Signaling Technology Cat. No. 9733T) antibodies. Secondary antibody incubation was performed with Guinea Pig anti-Rabbit IgG, (Antibodies-Online Cat. No. ABIN101961) for 30 min at RT with Digitonin150 Buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin). Beads were washed twice with Digitonin150 Buffer and incubated with pAG-Tn5 (EpiCypher Cat No. 15-1017) for 1hr in Digitonin300 Buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin). Beads were washed twice with Digitonin300 Buffer and tagmentation was performed at 37ºC for 1hr with Tagmentation Buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin, 10 mM MgCl2). Beads were washed with TAPS Buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA) and the tagmentation reaction was quenched with SDS Release Buffer (10 mM TAPS, pH 8.5, 0.1% SDS). Tagmented chromatin fragments were released by incubation for 1hr at 58ºC. SDS Quench Buffer (0.67% Triton-X 100) was added to the samples and library amplification was performed. We prepared unique index Nextera-compatible libraries with custom-designed index primers. PCR was performed with Non-hot Start Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific™ Cat No. F530S) for 14 cycles. Final DNA library fragment purification was performed with SPRI beads (ratio of 1.3 μl of SPRI beads to 1 μl of PCR product) and washed twice with 80% ethanol. The final library was resuspended in 0.1x TE buffer. CUT&Tag customized Nextera library