title: GSM3137179: INPUT_HTA11-Flag_17C_ZT0_rep2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Preparation the ChIP experiment was performed as described by Gendrel et al. (2002) with minor modifications. Plant material was immediately cross-linked when collected using 1% formaldehyde for 15min under vacuum. Chromatin was extracted from 1 g of cross-linked materials. For  MNAse-seq and CHIP-seq of: HTA11::HTA11-Flag, HTA11::HTA11-FLAG dek3-2, HTA11::HTA11-FLAG 35S::DEK3-CFP double mutant lines and H3.3-GFP, 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. MNase digestion was performed to obtain a mono-nucleosome resolution as MNase preferentially digests nucleosome-free DNA and the linker regions, whereas sequences bound by nucleosomes are relatively protected from the digestion. For MNAseseq samples have been used for library preparation directly after digestion. For DEK3::DEK3-CFP and 35S::DEK3-CFP CHIP-seq, 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 1 20mM Tris-HCl (pH8), 150mM NaCl, 2mM EDTA, 1% triton X-100 and 1X protease inhibitor cocktail. ChIP were performed for HTA11::HTA11-Flag using FLAG M2 magnetic beads (Sigma, M8823) and for DEK3::DEK3-CFP, 35S::DEK3-CFP, H3.3-GFP using anti GFP antibody (Abcam, ab290) coupled to a 1/1 mix of protein-A and protein-G Dynabeads (life technologies, 10001D and 10003D). All samples have been subsequently washed 2 times with low salt buffer (20mM Tris buffer (pH=8), 150mM NaCl, 0.1%SDS, 1% Triton) twice with high salt buffer (20mM Tris buffer (pH=8), 500mM NaCl, 0.1%SDS, 1% Triton), once with LiCl buffer (10mM Tris buffer (pH=8), 250mM LiCl, 1%Sodium deoxycholate, 1% NP40) and twice with TE buffer (10mM Tris buffer (pH=8), 1mM EDTA). The elution was done for anti-Flag ChIP with 100ng/ul of 3XFLAG in TE buffer and for anti-GFP ChIP with elution buffer (1% SDS, 0.1M NaHCO3). DNA was extracted using AMPure beads using a ratio of 2.3X as described previously. MNAseseq and CHIPseq libraries have been prepared in house using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®  (#E7645). The libraries were sequenced using paired-end 75bp on NextSeq500 on site. CHIP 35S::DEK3-CFP, CHIP HTR5::HTR5-GFP and HTR13::HTR13-GFP libraries have been prepared using the TruSeq ChIP library preparation kit (IP-202-1012). CHIP HTR5::HTR5-GFP and HTR13::HTR13-GFP libraries were sequenced on a NextSeq 500 using PE 75bp. CHIP 35S::DEK3-CFP libraries were sequenced using on a NextSeq 500 SE 75bp.
attributes.tag: GEO Accession
attributes.value: GSM3137179