GSM5009176: MM468_DMSO2_day67_H3K27me3 [bulk ChIP-seq]; Homo sapiens; ChIP-Seq
Sequential ChIP-seq:The first ChIP experiments were performed as described previously (Grosselin et al.,  2018) on 30e6 cells using anti-H3K27me3 (Cell signaling, Ref: #9733) or anti-H3 (Cell signaling, Ref: #4620) antibodies. After the washes, samples were eluted twice at 37°C for 15 min under agitation in the first ChIP elution buffer (50mM Tris-Hcl pH8, 5mM EDTA, 20mM DTT, 1%SDS)(Desvoyes et al., 2018). Samples were diluted 10 times to reduce SDS and DTT concentration. 10% of the eluted chromatin were kept as primary ChIP. The second ChIP was performed overnight on the rest of the chromatin using anti-H3K27me3, anti-H3K4me3 (Cell signaling, Ref: #9751) or anti-IgG (Cell signaling, Ref: #3900) antibodies. After the washes, samples were eluted twice at 65°C for 15 min under agitation in the ReChIP elution buffer (0,1M NaHCO3 and 1% SDS)(Desvoyes et al., 2018). As a positive control, we first immunoprecipitated H3 and the H3K27me3 and as a negative control, we first immunoprecipitated H3K27me3 followed by IgG immunoprecipitation.  manufacturer's instructions.   Bulk ChIP-seq : ChIP experiments were performed as previously described19 on 3x106 MDA-MB-468 cells (DMSO-D67-#2, DMSO-D77-#3, DMSO-D113-#4, 5-FU-D67-#2, 5-FU-D77-#3, 5-FU-D113-#4) using an anti-H3K27me3 antibody (Cell Signaling Technology, Ref: 9733 - C36B11).   Chromatin Indexing : Chromatin isolation, indexing, immunoprecipitation and library preparation was adapted from48. Briefly, 50,000 MDA-MB-468 were lysed and digested with MNase for 20min at 37°C in the following buffer: 46mM Tris-HCl pH 7.4, 0.154M NaCl, 0.1% Triton, 0.1% NaDoc, 4.65mM CaCl2, 0.47x Protease Inhibitor Cocktail (Roche, Ref: 11873580001) and 0.09u/uL MNase (Thermo Scientific, Ref: EN0181). Fragmented nucleosomes were then ligated for at least 24h at 16°C to double-stranded barcoded adapters containing 8bp barcodes to combine samples:  Pac1-T7-Read2-8bpBarcode-linker-Pac1 (Supplementary Table 1). Next, 5 indexed chromatin samples (DMSO, 5-FU, UNC, 5-FU + UNC, GSK-J4) were pooled, each containing a different 8-bp barcode, to perform anti-H3K27me3 ChIP (Cell Signaling, Ref: 9733 - C36B11) on 250,000 cells in total in each pool. Sequential ChIP-seq : After reverse crosslinking and DNA clean-up, 3 to 15ng of immunoprecipated DNA were used to prepare the sequencing libraries using the NEBNext Ultra II DNA Library Prep Kit (NEB, Ref: E7645S) according to theLibraries were sequenced on a NovaSeq 6000 (Illumina) in SE100 mode.  Bulk ChIP-seq : Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, Ref: E7645S) according to the manufacturer's instructions.  Libraries were sequenced on a NovaSeq 6000 (Illumina) in SE50 mode.  Chromatin Indexing : ChIP and DNA amplification was carried out as for scChIP-seq19 and a sequencing library was produced for both IP and input pools, sequenced on NovaSeq 6000 (Illumina) in PE100 mode.