p30-GMP-blast cells, from pool of Biological repl_I_II_III, CEBPA ChIP
strain/background
Lp30
tissue
tertiary transplanted leukemia, whole bone marrow, cKIT enriched, FACs isolated
sorted cell population
GMP
input dna amount
100pg
chip antibody
CEBPA
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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, 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). A proprietary protocol for small-scale ChIP-seq DNA amplification was used, based on further refining of the standard Illumina amplification and addition of bacterial carrier DNA. See associated publication for detailed protocols.