Modified from (Birch-Machin et al., 2005)
Main steps:
- Embryo collection and fixation
The aim of the method is to isolate fixed chromatin from Drosophila embryos. Essentially the chromatin is fixed, then nuclei are purified, nuclei are lysed and the chromatin is fragmented by sonciation. 1.5g of embryos are collected for each chromatin prep. This is usually enough to do at least 5 ChIP reactions.
- Chromatin prep from frozen fixed embryos with the bioruptor sonicator
- Chromatin IP using Protein A beads
- Blunting of IP-ed DNA: Optional, though advisable
- Purification of genomic DNA used as a reference in hybridizations
- Linker ligation + PCR amplification of chromatin IP eluates
- Quantitative real-time PCR verification of chromatin immunoprecipitation results
- Direct Klenow Labeling using BioPrime DNA Labling System
- Hybridization of Microarrays in Corning hybridization chambers