Sample Collection and INTACT Purification of PCC Nuclei

To evaluate the enrichment of PCC-specific nuclei after INTACT, ProSUC2:RedNTF ProUBQ10:BirA seeds were sown directly on soil and germinated in LD. Leaf material from approximately sixty 24-d-old plants per biological replicate was collected, frozen immediately in liquid nitrogen, and stored at −80°C until further use. A quantity of 1.5 g of the collected material was used for INTACT as described in You et al. (2017). Minor modifications compared with the original protocol were the addition of 5 mM dithiothreitol to the nuclear purification buffer and omission of bovine serum albumin during the purification.

The SD to LD shift experiments were performed with three independent biological replicates. For each biological replicate, ∼500 plants were grown in SD, and the 0 LD, 1 LD, 2 LD, and 3 LD samples were collected on the 21st d in SD and 1, 2, and 3 d after the shift to LD. For each sample, shoot material from ∼120 plants was collected and immediately frozen in 50-mL falcon tubes suspended in liquid nitrogen. The samples were collected at ZT 6 to 7 and samples were stored at −80°C before INTACT purification. The INTACT experiments were performed as described in You et al. (2017), with ∼1/3 of the ground tissue used for isolating nonfixed nuclei for transcriptome analysis by the RNA-seq method and the remaining ∼2/3 of the ground tissue used for isolating fixed nuclei and subsequently used to analyze histone modifications by the ChIP-seq method. The purity and yield of intact nuclei were assessed by microscopy and nuclear samples were stored at −80°C before further experiments.