GSM3354817: Mouse_ChIP_II_P30_GMP_H3K4ME1; Mus musculus; ChIP-Seq
Cells were lysed as described previously (Jakobsen JS, Waage J, Rapin N, Bisgaard HC, Larsen FS, Porse BT. Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries. Genome Res. 2013;23(4):592-603.) in 300 μl lysis buffer,12 applying up/down pipetting 10 times using a 100 ul tip low retention tip. Samples were sonicated using a Bioruptor sonicator plus for 30 cycles, 15/30 seconds on/off, high setting, and debris pelleted by centrifuging cold at 14,000g for 10 minutes. Fragmentation of chromatin was evaluated on extracted DNA using either: 1) a c-Kit enriched 500,000 cell sample processed in parallel (one of six tubes sonicated simultaneously) and agarose gel electrophoresis or, 2) direct sample size inspection of a 20 ul aliquot using a Bioanalyzer. ChIP was performed essentially as described previously (Sandmann T, Jakobsen JS, Furlong EE. ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos. Nat Protoc. 2006;1(6):2839-2855) with ca. 125.000 cells for histone mark and ca. 250.000 cells for CEBPA ChIP. Quanta of used antibodies (CEBPA, Santa Cruz sc-61, lot#J0407, 0.2 ug; H3K27me3, Cell signaling #9733S, lot#2, 1 ul; H3K4me3, Cell signaling #9751S,  lot#2, 1 ul) and protein A beads (Sigma, cat#P9424 , 10 ul 50%/50% beads/RIPA-low salt (140 mM)) were optimized for low input amounts, using siliconized tubes (Biozym, cat#1267-2970). NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (NEB #6200) or NEBNext Ultra I DNA Library Prep Kit for Illumina (NEB #7370), using bacterial carrier DNA as required, see: 'Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq', Jakobsen et al., BMC Genomics 2015