title: GSM2550651: WUS-GR ChIP-Seq1; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: ChIP assays were performed from 3 g of fresh weight each as previously described (Gendrel et al., 2005, and Pfeiffer et al., 2016) with modifications to tissue fixation and nuclei preparation. Plant material was fixed in 100mM sodium phosphate buffer, pH 7.0, 50mM NaCl, 100mM sucrose, and 0.33% formaldehyde for three times 10 min under vacuum. Nuclear extracts were prepared according to the INTACT protocol (Deal and Henikoff, 2011) and chromatin sheared according to Fan et al. (2008). Cleared nuclear extracts were split in two and incubated each with 20 μl of RFP-Trap_A (ChromoTek) for 4 h over night for immunoprecipitation. MinElute Reaction Cleanup Kit columns (Qiagen) were used for purification of the DNA fragments. Enrichment of specific DNA fragments was validated by qPCR at the ARR7 promoter region (Leibfried et al., 2005) by comparing immunoprecipitated DNA of WUS-GR and control samples. Two independent sequencing libraries were generated for the using pooled DNA from 9 individual ChIP preparations. Libraries were prepared for Illumina Sequencing using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (E6240) with the NEBNext Multiplex Oligos for Illumina (E7335 and E7500). Libraries were quantified using the Qubit Fluorometer from Invitrogen, and their size determined using the DNA 1000 Assay on the BioAnalyzer. Sequencing was performed on the Illumina HiSeq 2000 Instrument 50 base SE (plus index read).
attributes.tag: GEO Accession
attributes.value: GSM2550651