Multi-layer dosage compensation of the avian Z chromosome (MINUTE-ChIP)
Multi-layer dosage compensation of the avian Z chromosome (MINUTE-ChIP)
Pellets of 1 million chicken embryonic fibroblast cells were collected by centrifugation at 300g for 5min. To eliminate serum-containing medium residues, the pellets were washed 3 times in 1x PBS before a final centrifugation at 300g for 5min. Finally, the supernatant was completely removed without disturbing the cell pellets and the samples were placed in -80°C until further processing. Triplicate pellets of 10^6 cells were collected for all conditions, flash frozen and stored at −80 °C before use.  Immunoprecipitation was performed using the EpiFinder™ Genome kit (Epigenica, EpGe001) according to the manufacturer's instructions. In brief, native frozen cell pellets were lysed and MNase digested to mono- to tri-nucleosome fragments and ligated with double-stranded DNA adaptors in a one-pot reaction.  Barcoded samples were then pooled and aliquoted into individual ChIP reactions with Protein A (Dynabeads; Thermofisher) for the following antibodies: H3K4me3 [Millipore; 04-745], H3K27ac [Active Motif; 39034], H3K9ac [Active Motif; 39137-AF], H4K16ac [Millipore; 07-329]. Upon incubation overnight with rotation  at 4 °C and washing steps, ChIP DNA was isolated and set up in sequential reactions of adaptor fill-in, in vitro transcription, RNA 3′ adapter ligation, reverse transcription and PCR amplification to generate final libraries for each ChIP.