title: GSM7434747: BS-seq nrpe1 spt6l rep1; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: plants were crosslinked with 1% formaldehyde for 20 min under vacuum at room temperature and quenched with 0.125 M glycine under vacuum for an additional 5 min. Approximately 500 milligrams of tissues were used for each ChIP assay. The chromatin was sonicated using a Covaris M220 sonicator (peak power: 75; duty factor: 20; cycles/burst: 200) for 210 s at 6°C. The lysates were centrifuged at 18,000 g for 10 min at 4°C to remove debris. 10 µL of supernatant was used as the input sample, whereas the remaining supernatant was kept for the downstream procedure. 10 µL of Dynabeads Protein A (Cat. 10002D, ThermoFisher SCIENTIFIC) were added to each sample to prewash for 1 h at 4°C with gentle rotating. ChIP was carried out using antibodies as follows: anti-GFP (Cat. ab290, Abcam) and anti-Pol II CTD repeat YSPTSPS (Cat. ab817, Abcam). 40 µL of Dynabeads Protein A were mixed with 4 µL of antibodies and incubated for approximately 6 h on a rotator at 4°C to form antibody-bead complexes. Finally, ChIP-DNA were recovered by Phenol/chloroform extraction. Approximately 400 milligrams of seedlings were used per sample. Additionally, equal amounts of spike-in nuclei extracted from Saccharomyces cerevisiae were added to each sample before MNase treatment, which worked as the internal control among samples. For smRNA sequencing, total RNA was extracted from 10-day-old seedlings using TRIzol (Invitrogen, 15596-018). RNA samples were separated on a PAGE gel, and the 18- to 30-nt fraction of the gel was cut for small RNA purification. Library preparation and sequencing were performed using Illumina reagents according to the manufacturer's instructions. For the bisulfite sequencing, the DNA was extracted from 10-day old seedlingds and directly sent to the NovoGene for whole-genome bisulfite sequencing (WGBS). For RNA immunoprecipitation followed sequencing (RIP-seq), ChIP libraries were prepared by using the NEBNext® Ultra™ DNA Library Prep Kit (E7370S) following the manufacturer's instructions, and subsequently used for Illumina