Figure 6
10,000 cell histone mark and transcription factor ChIP-seq. (A and B) ChIP using 10,000 sorted GMP-blasts as input. Our protocol (Jakobsen) with and without mRNA/Histone carrier, compared to the Zwart protocol. qPCR assessment of enrichment ratios, n = 4-6, error bars show SD. *** = P < 0.001, T-test, Holm-Sidak correction for multiple testing. (A) H3K4me3-ChIP, ratios are amplicon Smc4 versus the negative Sfi3. (B) CEBPA-ChIP, ratios are amplicon Ptgs2 versus the negative Sfi3. (C) H3K4me3 and CEBPA ChIP-seq profiles across a region spanning a 130 kb region on chromosome 2, using 375,000 (H3K4me3-1) or 10,000 (H3K4me3-10 K) cells as input (two upper tracks) and CEBPA ChIP-seq profiles of using 250,000 (CEBPA-1) or 10,000 (CEBPA-10 K) cells as input (two lower tracks). (D) Quantitative comparisons of mean read coverage of H3K4me3-1 versus H3K4me3-10 K (left panel) and CEBPA-1 versus CEBPA-10 K (right panel). All settings as described above. (E) Peak overlaps between ALPHA-1 and ALPHA-2, CEBPA-6 and CEBPA-10 K. MACS2 peaks were ranked for FDR (q-values) and the CEBPA-1 top 1% set tested against peaks scoring FDR < 10−5, in the FDR top 1% or in top 10,000. (F) Schematic bar diagram depicting summed NGS-QC (Next Generation Sequencing – Quality Check) score for each of the listed small-scale ChIP-seq data sets. Listed is number of cells used as input material, source of material (TC: tissue culture cells, IV: in vivo cells) and antibody target (HM: histone mark, TF: transcription factor). The ChIP-DNA fraction column denotes amplification of a fraction of DNA from a higher-cell-number ChIP corresponding to the indicated number of cells. The carrier column indicates use of mRNA/Histone as carrier during the ChIP procedure (Y = yes). All scores represent best replicate. NGS-QC sum score is calculated as a sum of quartile scores (best = 4, worst = 1) for read sub-sampling of 90, 70 and 50% of total mapped reads.