|Public on May 12, 2009
|1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a collection of third instar larvae (L3), new plates are added after a 2 hours pre-egglaying at 9 am and flies are allowed to lay eggs for 24 hours. Plates are then kept at 25C for another 48 hours. 3. L3 larvae are collected at 8.45 am using a filter mesh and a brush. L3 larvae are the larvae not going through the first mesh. They are rinsed with EWB and cross-linked at 9 am in 1.8% formaldehyde at room temperature for 15 min.
|Drosophila samples are crushed in presence of 1.8% of formaldehyde for 15 minutes. After washing, the material is sonicated for 15 minutes to solubilize the chromatin. After centrifugation, the supernatant containing soluble chromatin is collected for IPs. Specific antibodies are added to the chromatin extract while one aliquot will be treated for DNA extraction corresponding to the reference samples. ChIP are carried overnight at room temperature. Protein A Sepharose beads are used to pull down the chromatin bound antibodies. After washes and finally elution, the DNA corresponding to the IPed material is extracted and purified. The DNA is directly used for Solexa library preparation.
|Illumina Genome Analyzer
|Reads were aligned to the flybase BDGPv5 reference genome, and the fragment count at any given position (10-bp resolution) was estimated as the number of uniquely aligned reads oriented towards it and within 300 bp. Enriched intervals were identified by comparison of the mean fragment count in 1-kb windows against a sample-specific expected distribution estimated by randomization (H3K4me3, H3K27me3), or using a supervised Hidden Markov Model (H3K36me3, H3K9me3, H4K20me3).
|May 07, 2009
|Last update date
|May 15, 2019
|Kevin P. White
|University of Chicago
|Institute for Genomics and Systems Biology
|900 E. 57th STR. 10th FL.
|Drosophila at different time points of development: ChIP-chip, ChIP-seq, RNA-seq
|Genome-wide maps of chromatin state in staged Drosophila embryos, ChIP-seq