Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: ChIP from ED or whole 14-16h embryos were performed in duplicate as previously described in Loubiere et al., 2016. Briefly, ~200 ED from wandering larvae were used per replicate for PTM ChIP-Seq and ~500 for PSC and SU(Z)12 ChIP-Seq. For each replicate of PTM ChIP-Seq from 14-16h embryos, 2h collections were performed at 25°C with about 500 flies. When necessary, several batches of dissection/collection were snap-frozen in liquid nitrogen and stored at -80°C in order to collect enough material. Samples were then transferred into a tight Tenbrock and cross-linked for 15min at RT using 1.8% formaldehyde and continuous homogenization before being lysed on ice for 2h with 1% SDS Lysis Buffer 2 (see Loubiere et al., 2016). Chromatin was sonicated using a Bioruptor (Diagenode) for 18 cycles (settings 30s ON, 30s OFF, high power) and the quality of the sonication was checked on a 1.5% agarose gel (~300bp fragments). Then, samples were pre-cleared O/N using 15µL of protein A Dynabeads (ThermoFisher Scientific, 10001D) at 4°C and an aliquot was kept apart to constitute the INPUT. Antibodies were added (see Supplementary Table 7) 4h prior to the addition of 30µL of protein A dynabeads and O/N incubation at 4°C. Finally, beads were washed, and the precipitated chromatin was eluted before O/N decrosslinking at 65°C, Proteinase K treatment, Phenol/Chloroform purification and O/N ethanol precipitation. Libraries were prepared using the TruSeq ChIP Sample Preparation kit from Illumina, following manufacturers' instructions.