GSM6751468
Chromatin or RNA was extracted from 14-days-old seedlingss, using standard protocols. For RNA-seq, library construction and deep sequencing were performed by Novogene (Beijing, China) using Illumina NovaSeq 6000 system to produce 150-bp paired-end reads. For ChIP-seq, crosslinked materials were ground into fine power with liquid nitrogen and resuspended in ChIP Lysis Buffer1 (CLB1: 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% Glycerol, 1x inhibitor Cocktail, 0.035% 2-Mercaptoethanol) and incubated for 60 minutes with rotation at 4°C. After incubation, collect the nucleus after filtering the mixture through 40-um strainer, centrifuging at 3000 g for 30 minutes at 4°C in a swinging bucket rotor and removing the supernatant. The nucleus was washed twice with ChIP Lysis Buffer2 (CLB2: 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% Glycerol, 1x inhibitor Cocktail). DNA was sheared by sonication to approximately 300–500-bp fragments. After centrifugation (10 minutes at 13,000 rpm), the supernatant was precleared with 40 μl salmon sperm (SS) DNA/Protein A agarose for 60 minutes at 4°C. After 2 minutes of centrifugation at 500 g, the supernatant was transferred to a siliconized tube, and 10 μl of the appropriate antibody (H3 trimethyl-Lys 27 (ABclonal, A2363), H3 trimethyl-Lys 4 (Abcam, Cambridge, England), and H3 acetyl-Lys 9 (Millipore)) was added. After incubation overnight with rotation, 40 μl SS DNA/Protein A agarose was added and incubation continued for 1 hr. The agarose beads were then washed with 1 ml of each of the following: Low salt buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA), High salt buffer (50 mM HEPES [pH 7.5], 635 mM NaCl, 1 mM EDTA), LiCl wash buffer (0.25M LiCl, 0.5% NP-40, Tris-HCl [pH 8], 1 mM EDTA), and 1× TE (10 mM Tris-HCl [pH 8], 1 mM EDTA). The immunocomplexes were eluted from the beads with 400 μl 1% SDS, 0.1 M NaHCO3. A total of 20 μl 5 M NaCl was then added to each tube, and crosslinks were reversed by incubation at 65°C for 5–6 hours. Residual protein was degraded by the addition of 20 μg Prot K (in 10 mM EDTA and 40 mM Tris [pH 8.0]) at 45°C for 1 hour, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Pellets were washed with 70% EtOH and resuspended in 30 μl TE buffer. More than 10 ng ChIP DNA was used to prepare each sequencing sample. Libraries were constructed and sequenced by Novogene (Beijing, China). The libraries were sequenced with the Illumina NovaSeq 6000 system to produce 150-bp paired-end reads. For spike-in ChIP-seq, Arabidopsis chromatin was added into wheat chromatin before immunoprecipitation. Briefly, before performing immunoprecipitation, 40 ul of chromatin was reverse crosslinked, proteinase K digested followed by DNA purification. The concentration of purified DNA was determined using Qubit dsDNA High-Sensitivity Assays (Invitrogen). Meanwhile, chromatins from Arabidopsis were also isolated and determined the concentration with the same method. One percent of Arabidopsis chromatins were mixed with different Wheat samples before immunoprecipitation. The rest procedures are the same as the previous description. For nascent RNA (chromatin-bound RNA, CB RNA) library preparation, the chromatin RNA extraction was completed as previously described54. After degrading genomic DNA by TURBO DNase (Life Technologies), CB RNA was subjected to rRNA depletion using a riboPOOL kit (siTOOLs Biotech, PanPlant-10 nmol) and polyA RNA removal by oligo(dT) beads (NEB, S1419). Both CB RNA and polyA RNA were transformed into cDNA libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765) and sequenced on an Illumina NovaSeq platform.