title: GSM6734106: wt_4h_FLA_DRIP_rep2; Arabidopsis thaliana; OTHER
library_name: GSM6734106
library_construction_protocol: ssDRIP-seq: Nuclei were isolated from Arabidopsis plants with Honda buffer, followed by SDS/proteinase K treatment at 37°C overnight. Genome DNA was extracted by the phenol-chloroform method and precipitated with isopropanol. DNA fragmentation was performed overnight using endonucleases (MboI, MseI, NlaIII, DdeI); the negative control was treated with RNase H (New England Biolabs) overnight. DRIP was performed as described in Xu et al., 2017 and Sun et al., 2013. Briefly, 3.5 ug of DNA was bound with 10 ug of S9.6 antibody in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4˚C. 50 ul Protein G sepharose beads were added for 4 hours. Bound beads were washed 4 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, Proteinase K) for 45 min at 55˚C. DNA was purified and sonoicated to 150 bp fragments. pNET-seq was performed as described (Zhu et al., 2019). In brief, nuclei were extracted from 12 days old seedlings using percoll-gradient (30%-80%) and washed with 1X transcription buffer (2 M Hexylene glycol. 20mM PIPES-KOH [PH 7], 10mM MgCl2, 5mM β-mercaptoethanol) and 1x nuclei storage buffer (50mM Tris-HCL [PH7.2], 5mM mgCl2, 5mM KCl, 1mM MnCl2, 5mM DTT, 50% glycerol), respectively. After that nuclei were resuspended in 1x reaction buffer (1mM ATP, 1mM GTP, 1mM CTP, 1mM Br-UTP, 4U RNasin, 1% sarcosyl) and incubated at 30C for 5 minutes. The reaction was stopped with TRIzol and nuclear RNA was precipitated with 1/10 vol 5M NH4AC, 3 vol Ethanol and 1μl GlycoBlue. RNA was then treated with 5μl 1M NaOH for 40min on ice, and the reaction was stopped with 25μl 1M Tris-HCl pH7.0. After RNA purification with Bio-spin 30 Column (Cat. #732-6250/6251) and DNase treatment with RQ1 RNase-free DNase (Promega), RNA de-phosphorylation was performed with antarctic phosphatase (NEB, B0289S), and incubated with pre-blocked Anti-BrdU argarose beads (Santa Cruz Biotech) and the reaction mixture was rotated for 1 hr at 4C. Beads were then was with Low salt (0.2x SSPE, 1mM EDTA, 0.05% tween-20), high salt (0.5x SSPE, 1mM EDTA, 0.05% tween-20, 150mM NaCl) and TET buffers (10mM Tris-HCL [PH 8], 0.1mM EDTA, 0.05% Tween-20), respectively. RNA was then eluted from beads with elution buffer (20mM DTT, 300mM NaCl, 5mM Tris [PH 7.5], 1mM EDTA, 0.10% SDS), and precipitated with 1/10 vol 5M NH4AC, 3 vol Ethanol and 1μl GlycoBlue. RNA was then end-repaired with T4 PNK (NEB, M0201L) and RNAsin Plus (Promega, N2611) and precipitated with 1/10 vol 5M NH4AC, 3 vol Ethanol and 1μl GlycoBlue. Ploy A tail was added to end-repaired DNA with poly-A polymerase (NEB, B0276S), and reverse transcription was performed with SSIII reverse transcriptase (Invitrogen). After that extra RT-primers and RNA were cleaned, cDNA was size selected (more than 120 bp) with page gel. Size selected cDNA was circularized with CircLigase (Epicentre) and relinearized with 4× relinearization supplement (100mM KCl, 2mM DTT) and Ape I (NEB, M0282S). Then relinearized DNA was run on page gel, and precipitated with 1/10 vol 5M NH4AC, 3 vol Ethanol and 1μl GlycoBlue. Size selected cDNA was finally amplified with 2x Kapa HiFi mixture (KK2602), using universal forward primer F and reverse index primer R. The amplified libraries were size selected, purified and precipitated with page gel. ChIP-seq: To gain the nucleosome resolution, native native H3K9me2, H3K27me1, H2A.W-FLAG, H2A.Z ChIP without crosslink and sonication was performed as described in (Bernatavichute et al., 2008), with some modifications. In brief, nuclei were extracted from 12 days old seedlings with Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES–KOH [pH 7.4], 10 mM MgCl2, 0.5% Triton X-100, 5 mM DTT, and protease inhibitors [Roche]). Nuclei were suspended in 1X MNAse buffer (50mM TriHCl pH 7.6, 5mM CaCl2, 0.1mM PMSF) containing 1X proteinase inhibitor cocktail (Roche). After RNase A (Thermo Scientific™) treatment at 37C for 30 minutes, chromatin was digested with MNase enzyme (NEB) for 6 minutes and the reaction was stopped with EDTA (10mM). Then, the nucleosomes were released with the addition of 0.005% SDS while rotating at 4C for 2-3 hours. After that the supernatant was separated and diluted with dilution buffer (0.1% triton, 50mM NaCl, 0.1mM PMSF, 17mM trisHCl, 3.33mM EDTA, 1x Cocktail inhibitor). Chromatin was pre-cleaned with Protein G beads, and immnoprecipitated with anti-H3K9me2 (Millipore),anti-H3K27me1 (Millipore), anti-FLAG (Sigma) and anti-H2A.Z antibodies at 4C overnight. The complexes were incubated with protein G beads (Millipore) at 4C for 3 hours with rotation, and washed three times with ChIP wash buffer having different concentration of NaCl in each wash (50mM TrisHCl pH 7.6, 10mM EDTA, 50/100/150 mM NaCl, 0.1mM PMSF, and 1x Cocktail inhibitor). Then DNA was eluted with elution buffer (0.1%SDS and 0.1M NaHCO3) at 65C for 15 minutes. Proteinase K treatment was performed at 55 C for 2 hour. DNA was recovered after phenol:chloroform extraction. ssDRIP-seq: DRIPed DNA was conducted to library construction using the VAHTS ssDNA Library Prep Kit (Vazyme, ND620). Bisulphite-seq: to use EpiArt DNA Methylation Library Kit for Illumina V3 (Vazyme, NE103) for library construction. ChIP-seq: ChIPed DNA was conducted to library construction using the TruePrep DNA Library Prep Kit V2 (Vazyme, TD502). pNET-seq:  NEXTflex TM small RNA-Seq Kit V3 was used to construct the libraries.