TAF-ChIP: an ultra-low input approach for genome-wide chromatin immunoprecipitation assay

(A) ROC curves of TAF-ChIP and CUT&RUN for H3K27Me3 in K562 cells. The ROC curves were plotted using as reference replicated peaks of the conventional ChIP-Seq ENCODE dataset selected at 5% FDR cutoff (downloaded from the ENCODE database). No FDR cutoff was used to define peaks for TAF-ChIP replicates and the CUT&RUN datasets with MACS2. Peaks were mapped to 5 kb non-overlapping genomic windows to calculate true-positive rate or recall, false-positive rate and precision for a changing P-Value threshold. Area under the curve (AUC) is indicated in the legend in decreasing order, and the * indicates the failure to faithfully calculate the AUC. (B) Precision-recall curve for TAF-ChIP and CUT&RUN datasets for H3K27Me3 in K562 cells. (C, D) ROC curves of TAF-ChIP and conventional ChIP-Seq in Drosophila NSCs. The ROC curves for H3K4Me3 (C) and H3K9me3 (D) were plotted using as reference peaks of the first conventional ChIP-Seq replicate selected at 5% FDR cutoff. No FDR cutoff was used to define peaks for TAF-ChIP replicates and the second conventional ChIP-Seq replicate. Peaks were mapped to 1 kb non-overlapping genomic windows to calculate true-positive rate or recall, false-positive rate, and precision. AUC is indicated in the legend in decreasing order. (E, F) Precision-recall curve for TAF-ChIP and conventional ChIP-Seq in Drosophila NSCs. Using same references and data as above, precision-recall curves were plotted for H3K4Me3 (E) and H3k9Me3 (F). (G) Comparison of the genomic window sets for Drosophila brain-derived wt NSCs analyzed for H3K4Me3 binding by TAF-ChIP (TAF_Wt), and Drosophila tumor-derived NSCs analyzed by TAF-ChIP or conventional ChIP-Seq (TAF_Tum and Conv). The TAF-ChIP samples are highlighted by a red rectangular box. The heat map indicates pairwise similarity according to the Jaccard index. Axes show results of hierarchical clustering. (H) The Jaccard index and hierarchical clustering, as described in (G), to compare H3K27Me3 binding in K562 cells. The comparison was performed for 100 cells TAF-ChIP samples (highlighted with a red rectangular box), to CUT&RUN method with 100, 3,000, and 6,000-cell samples, and to conventional ChIP-Seq (ENCODE) (12, 19).

(A, B) Genome browser track example of H3K27Me3 and H3K9Me3 (A and B, respectively) ChIP performed in 100 FACS sorted K562 cells with TAF-ChIP approach and corresponding K562 conventional ChIP-Seq datasets from the ENCODE project in duplicates, as indicated in the labels. The label below the tracks shows the gene model and the y-axis represents normalized read density in reads per million. The enriched regions are highlighted with shaded box. (C, D) Metagene profiles of H3K27Me3 and H3K9Me3 (C and D, respectively) with standard error to the mean for all the genes, −1,000 bp upstream of TSS and +1,000 bp downstream of transcription end sites (TES). Read counts per million of mapped reads is shown on the y-axis, whereas the x-axis depicts genomic coordinates. (E) Genomic distribution of annotated peaks obtained from the ENCODE datasets and TAF-ChIP (100 K562 cells), for indicated histone marks. Note the majority of H3K27Me3 and H3K9Me3 peaks are at the intergenic regions, consistent with the expectation. (F) Overlap between the peaks identified from the ENCODE and TAF-ChIP datasets, for the indicated histone modifications (see the Materials and Methods section for further details). (G) Average profile of TAF-ChIP and corresponding ENCODE ChIP-Seq centered at the peaks for the indicated histone modifications. The y-axis depicts average per base value into the peaks, whereas x-axis depicts genomic coordinates centered at the peaks. (H) Distributions of reads at gene locations of indicated histone modifications from ENCODE ChIP-Seq and TAF-ChIP method, centered at the peaks (−1 kb to +1 kb). Rows indicate all the peaks and are sorted by decreasing affinities in the ENCODE ChIP-Seq datasets. The color labels to the right indicate the level of enrichment.

(A, B) Genome browser track example of H3K4Me3 and H3K9Me3 ChIP (panel A and B, respectively) performed in FACS-sorted NSCs with conventional ChIP-Seq (1.2 million cells) and TAF-ChIP (1,000 cells), as indicated by the labels. The label below the tracks shows the gene model and the y-axis represents normalized read density in reads per million (rpm). (C) Metagene profiles of H3K4Me3 with standard error to the mean for all genes, −1,000 bp upstream of TSS and +1,000 bp downstream of transcription end sites (TESs). Log2-fold changes against input controls are shown on the y-axis, whereas the x-axis depicts genomic coordinates. (D) Metagene profiles of H3K9Me3 with standard error to the mean for enriched regions, −1,000 bp upstream and +1,000 bp downstream of peaks. Log2-fold changes against input control are shown on the y-axis, whereas the x-axis depicts genomic coordinates. (E) Distribution of annotated peaks obtained from conventional ChIP-Seq and TAF-ChIP, for indicated histone marks. Note that most H3K4Me3 and H3K9Me3 peaks are at the promoters and at the intergenic regions, respectively, consistent with the expectation. (F) Overlap between the peaks identified from conventional ChIP-Seq and TAF-ChIP datasets, for the indicated histone modifications. MACS2 software with identical parameters (see the Materials and Methods section for details) was used to identify the peaks against the respective input controls, and those present in both replicates were considered for the comparison. (G) Average profile of TAF-ChIP and conventional ChIP-Seq centered at the peaks for the indicated histone modifications. The y-axis depicts average per base value into the peaks, whereas the x-axis depicts genomic coordinates centered at the peaks. (H) Distributions of reads at gene locations of indicated histone modifications from conventional ChIP-Seq and TAF-ChIP. Rows indicate all the peaks and are sorted by decreasing affinities in the conventional ChIP-Seq datasets. The color labels to the right indicate the level of enrichment.