A1 (CRX1203717; CRA019323)
Rosette leaves were ground in liquid nitrogen into fine powder. Then, transfer 0.3 g powder into 2 mL tube and nuclei were isolated with 600 microL Buffer F. The homogenate was crosslinked with 1% formaldehyde for 20 min at room temperature and subsequently quenched in 125 mM glycine. Samples were centrifuged at 12000X g for 5 min and then discard the supernatant. The pellet was washed with 1 mL Buffer F for twice, and lysed in 600 microL Buffer S for 10 min in ice. The lysates were mixed with 400 microL Buffer F and the chromatin was sheared into 200-500 bp DNA fragments using Covaris S220. After centrifugation at 18000X g for 10 min, the supernatant was diluted 5-fold with Buffer F, and 10 microL Protein A/G beads were added to each sample to remove unspecific binding for 1 h at 4degree C with gentle rotating. Antibodies used were as follows: HA (CST, C29F4), HA (Abcam, ab9110), Pol || Ser5P (Abcam, ab5408), Pol || Ser2P (ABclonal, AP0749). Take the suitable amount Protein A/G beads (Vazyme, PB101), wash three times with 1 mL Buffer F, and incubate with 3-5 microg antibody for 4-6 h at 4degree C with gentle rotating. The precleaned chromatin was incubated with the antibody-beads complexes overnight at 4degree C with rotation. The beads were washed sequentially with Low salt buffer, High salt buffer, LiCl buffer, TE buffer. The protein-DNA complexes were eluted from beads with ChIP Elution buffer for 15 min at 65degree C. The supernatant was transferred to a new tube and then reverse cross-linked with 10% chelex resin (BIO-RAD, 1421253) for 10 min at 95degree C. Subsequently, the supernatant was digested with proteinase K and RNase for 1 h at 45degree C. DNA was purified by phenol: chloroform: isoamyl alcohol and used for qPCR or sequencing. QPCR was performed with SYBR Green Master Mix using CFX96 Real-Time System (Bio-Rad). For sequencing, ChIP DNA libraries were constructed using Scale ssDNA-seq Lib Prep Kit for Illumina V2 (ABclonal, RK20228). All samples were sequenced on the Illumina platform by Kindstar Sequenon.