GSM8389267: ADF8_CUTtag_H2AZ_1h_rePi_rep2; Arabidopsis thaliana; OTHER
Biotin tagged nuclei from ~100 mg from root tissue 1 cm above the root tip were captured on 25 uL of Streptavidin M280 Dynabeads according to the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol. Bead bound nuclei for each timepoint in the starvation assay were counted in 1 mL of Nuclei Purification Buffer with 0.1% Triton X-100 (NPBt) and nuclei were split three ways to ~100,000 nuclei per sample in NPBt for three replicates. CUT&Tag was performed on these bead bound nuclei according to the Epicypher CUT&Tag Protocol v1.7 with the following notes. All buffers were prepared without Digitonin. Bead bound nuclei were resuspended directly from NPBt into 50 uL of Antibody150 Buffer containing 0.5 ug of anti-H2A.Z (Repression of Flowering in Arabidopsis Requires Activation of FLOWERING LOCUS C Expression by the Histone Variant H2A.Z Deal RB, Topp CN, McKinney EC, Meagher RB the Plant Cell Online, 2007) and incubated at 4°C overnight. Guinea pig anti-rabbit secondary (0.5 ug, Sigma SAB3700889) was incubated for 1 hr at room temperature in 50 uL Wash150 Buffer. Tagmented libraries were amplified using 17 PCR cycles.