On column dnase 1 treatment

cKIT-enriched BM cells were isolated from young (2 months old) C57BL/6.SJL female mice and split into four groups (approximately 900,000 cells per sample). Untreated cells were kept on ice throughout the whole procedure. Cells from remaining samples were spun down, resuspended in 2% PFA in PBS and incubated at room temperature for 15 min. Cells were spun down at 400g for 5 min after adding wash buffer (PBS supplemented with 1% BSA and 40 U ml⁻¹ SUPERase•In, Invitrogen) and were subsequently resuspended in the wash buffer. For reverse crosslinking, cells were incubated either at room temperature for 5 min in wash buffer supplemented with 125 mM glycine (Serva) or at 56 °C for 60 min in wash buffer supplemented with 40 U ml⁻¹ proteinase K (Roche), followed by incubation on ice for at least 5 min. RNA was isolated using the Direct-zol RNA MicroPrep Kit with on-column DNase I treatment (Zymo Research). RNA integrity was measured using the RNA 6000 Pico Kit and a 2100 Bioanalyzer instrument (Agilent Technologies).

Total RNA from seedlings of the indicated genotypes and growth conditions was isolated using a Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO) or Quick-RNA MiniPrep Kit with on-column DNase I treatment (Zymo Research, Irvine, CA). cDNA was synthesized from total RNA using a Superscript II First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. Oligo(dT) primers were used for the analysis of nuclear gene expression, and a mixture of plastidial-gene-specific primers was used for the analysis of plastidial genes. qRT-PCR was performed with FastStart Universal SYBR Green Master Mix on a LightCycler 96 System (Roche, Basel, Switzerland). The mRNA level of gene-of-interest was normalized to that of PP2A. Primers for qRT-PCR and cDNA synthesis are listed in Supplementary Tables 1 and 2.