title: GSM2092799: HTA11_ChIP-seq_17c_1hr_37Exp; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: ChIP was performed as described (Gendrel et al., 2002) with minor modifications. In brief, chromatin was extracted from 1 g of crosslinked materials. For HTA11 (a highly expressed H2A.Z gene) and H3, the chromatin was resuspended in MNase digestion buffer (20mM Tris-HCl [pH8], 50mM NaCl, 1mM DTT, 0.5% NP-40, 1mM CaCl2, 0.5mM phenylmethylsulfonyl fluoride (PMSF) and 1X protease inhibitor cocktail [Roche]), and digested with 0.4U/ml of Micrococcal nuclease (MNase, Sigma, N3755) for 15 min. Digestion was stopped with 5 mM EDTA. For HSF1, chromatin was fragmented by sonication using a bioruptor (Diagenode) in lysis buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA [pH 8], 0.1% deoxycholate, and 1X protease inhibitor cocktail). All ChIPs were performed in a buffer containing 20mM Tris-HCl (pH8), 150mM NaCl, 2mM EDTA, 1% triton X-100 and 1X protease inhibitor cocktail. ChIP were performed for HTA11 and HSF1 using FLAG M2 magnetic beads (Sigma, M8823) and for H3 using H3 antibody (Abcam, ab1791) coupled to a 1/1 mix of protein-A and protein-G Dynabeads (life technologies, 10001D and 10003D). All ChIPs were performed in a buffer containing 10 mM Tris-HCl [pH 8.0], 5 mM EDTA [pH 8.0], 150 mM NaCl, 1% Triton X-100, and 1× protease inhibitor cocktail. The elution was done for HTA11 and HSF1 ChIP with 100ng/ul of 3XFLAG in TE buffer and for H3 ChIP with elution buffer (1% SDS, 0.1M NaHCO3). The efficiency of ChIP materials was assessed by qPCR in prior to in-house library preparation, using the TruSeq ChIP sample preparation kit (Illumina, IP-202-1012) following the manufacturer’s instructions. The libraries were sequenced on HiSeq2000 by MPI Tubingen and BGI using paired-end 100bp length reads, and on NextSeq500 at the Cambridge Genomic Services sequencing facility, Department of Pathology, University of Cambridge and at the Sainsbury Laboratory Cambridge University using paired-end 75 bp length reads .