title: GSM5684005: fwa_ATACseq_rep-1; Arabidopsis thaliana; ATAC-seq
library_construction_protocol: ChIP seq: Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer's instructions. BS seeq: 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). Libraries were prepared with the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer's instructions. RNA seq: 1 ug of total RNA was used for library preparation with TruSeq Stranded mRNA kit (Illumina). Libraries were sequenced on HiSeq 2500 or NovaSeq 6000 (Illumina). All Arabidopsis plants used in this study are in Columbia (Col-0) ecotype and were grown at 22°C in LD (16 hours light, 8 hours dark) conditions. ATAC-seq: The nuclei collection process from inflorescence and meristem tissues is as described previously. Freshly isolated nuclei were used for ATAC-seq as described elsewhere. Inflorescence tissues were collected for extraction of nuclei as follows. About 5 g of inflorescence tissue was collected and immediately transferred into ice-cold grinding buffer (300 mM sucrose, 20 mM Tris pH 8, 5 mM MgCl2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, and 35% glycerol). The samples were ground with Omni International General Laboratory Homogenizer at 4°C and then filtered through a two-layer Miracloth and a 40-µm nylon mesh Cell Strainer (Fisher). Samples were spin filtered for 10 min at 3,000 g, the supernatant was discarded, and the pellet was resuspended with 25 ml of grinding buffer using a Dounce homogenizer. The wash step was performed twice in total, and nuclei were resuspended in 0.5 ml of freezing buffer (50 mM Tris pH 8, 5 mM MgCl2, 20% glycerol, and 5 mM β-mercaptoethanol). Nuclei were subjected to a transposition reaction with Tn5 (Illumina). For the transposition reaction, 25 µl of 2x DMF (66 mM Tris-acetate pH 7.8, 132 mM K-Acetate, 20 mM Mg-Acetate, and 32% DMF) was mixed with 2.5 µl Tn5 and 22.5 µl nuclei suspension at 37°C for 30 min. Transposed DNA fragments were purified with ChIP DNA Clean & Concentrator Kit (Zymo). Libraries were prepared with Phusion High-Fidelity DNA Polymerase (NEB) in a system containing 12.5 µl 2x Phusion, 1.25 µl 10 mM Ad1 primer, 1.25 µl 10 mM Ad2 primer, 4 µl ddH2O, and 6 µl purified transposed DNA fragments. The ATAC-seq libraries were sequenced on HiSeq 2500 platform (Illumina). Plants total RNAs were extracted with TRIzol and Direct-zol RNA Miniprep kit (Zymo, R2050). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina) following the manufacturer instructions. RNA-seq library preparation: Plants total RNAs were extracted with TRIzol and Direct-zol RNA Miniprep kit (Zymo, R2050). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina) following the manufacturer instructions. ChIP-seq library preparation: 10 grams of inflorescence and meristem tissues were used for Flag, HA, Pol II, H3, and H3K36me2/3 ChIP-seq. ChIP were performed as described previously60 with anti-FLAG M2 (Sigma), anti-HA (Roche), anti-Pol II (Ab26721, Abcam), anti-H3 (Ab1791, Abcam), anti-H3K36me2 (Ab9049, Abcam), and anti-H3K36me3 (Ab9050, Abcam) antibodies. Anti-NRPB1 antibody was raised in rabbits and further affinity-purified by ABClonal (China) using the peptide HEGDKKDKTGKKDASKDDK. To evaluate Pol II occupancy at both 5' end and 3' end of gene body, we selected anti-RNA Pol II CTD repeat YSPTSPS antibody which can capture Pol II signal at both regions (Ser5P and Ser2P). Libraries were prepared with NuGen Ovation Ultra Low System V2 kit following the manufacturer instructions. Whole genome bisulfite sequencing (BS-seq) library preparation: Leaf tissue was used as starting material for BS-seq libraries preparation. Genomic DNA was extracted and converted with bisulfite treatment with EpiTect Bisulfite Kit (Qiagen) following the manufacturer instructions. TSS-seq library preparation: TSS-seq was performed on 14 day old seedlings grown on MS plate following previous protocol. Heat stress was conducted by treating plants with 2 hours of 37°C. Five micrograms of total RNA were treated with DNase and CIP (NEB) to remove DNA and all non-capped RNA. Then 5' caps of capped RNA were removed with Cap-Clip (CellScript) and single-stranded rP5_RND adapter were ligated to 5'-ends with T4 RNA ligase 1 (NEB). Ligated RNAs were enriched and captured by oligo(dT) Dynabeads (Thermo Fisher Scientific). Enriched samples were fragmented for 5 mins at 80°C and first-strand cDNA was generated with SuperScript III (Invitrogen) and random primers. Second-strand cDNA was synthesized with Phusion High-Fidelity DNA Polymerase (NEB) and the BioNotI-P5-PET oligo, and captured by Dynabeads for end repairing with End Repair Enzyme Mix (NEB) and ligation with barcoded Illumina compatible adapter using T4 DNA Ligase (NEB). TSS-seq sequencing libraries were amplified and size selected for single-end sequencing with NovaSeq 6000 platform (Illumina). BS-PCR and McrBC assay for FWA tandem repeat: For BS-PCR of FWA tandem repeat, genomic DNA was extracted from leaf tissue of ASF1B-ZF T2 with CTAB-based method and converted using the EZ DNA Methylation-Lighting kit (ZYMO research). Methylation level of FWA promoter region and several control regions have been amplified with primers described previously46. HiSeq 2500 (Illumina) sequencing libraries were made from purified PCR products using a Kapa Hyper DNA Library Prep kit. For McrBC of FWA tandem repeat, genomic DNA was extracted from leaf tissue with CTAB-based method, and treated with PureLink RNase (Invitrogen) and then with McrBC (NEB) for 4 hours at 37°C. FWA tandem repeat region was quantified by qPCR. ATAC-seq; RNA-seq; ChIP-seq; WGBS; TSS-seq; BS-PCR;