ChIP-seq Validated Antibodies

A successful ChIP-seq experiment requires an antibody that recognizes the correct target protein in all sequence contexts across the entire genome. Good antibody performance in ChIP-qPCR does not necessarily mean the antibody will perform well for ChIP-seq, because ChIP-seq requires more extensive capture of the target protein across a large number of gene loci. To provide antibodies that are proven effective for ChIP-seq, CST validates recombinant rabbit monoclonal antibodies for ChIP-seq using the SimpleChIP^® Enzymatic and SimpleChIP^® Sonication Chromatin IP protocols followed by next-generation sequencing (NGS).

ChIP-seq Antibody Validation Steps

All ChIP-seq validated antibodies are first subjected to the ChIP-qPCR validation protocol.
Antibody sensitivity for ChIP-seq is then confirmed by analyzing the signal:noise ratio of target enrichment across the genome in antibody:input control comparisons. The antibody must provide an acceptable minimum number of defined enrichment peaks and a minimum signal:noise threshold compared to input chromatin.
For sequence-specific DNA-binding transcription factors, antibody specificity is determined by performing motif analysis of enriched chromatin fragments.
Antibody specificity is further determined by comparing enrichment across the genome using multiple antibodies against distinct target protein epitopes.
Antibody specificity is confirmed using antibodies against different subunits of a multiprotein complex.
Antibody specificity is further confirmed by comparing enrichment across the genome to published ChIP-seq data (ie, ENCODE) using additional antibodies for a given target protein.

Signal:Noise

Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP^® Enzymatic Plus Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows a specific binding of TCF4/TCF7L2 across c-MYC, a known target gene of TCF4/TCF7L2 (upper track), compared to Input control (lower track). The average signal:noise ratio across the c-MYC gene is 21.58.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP^® Enzymatic Plus Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. As shown in the figure, the average signal:noise ratio across the whole genome for this experiment, comparing the TCF4/TCF7L2 enrichment (blue line) to the Input control (red line), is 4.44.

Antibody Specificity for Transcription Factors

Chromatin immunoprecipitations were performed with cross-linked chromatin from A549 cells cultured in media with 5% charcoal-stripped FBS for 3 d and then treated with 100 nM dexamethasone for 1 hour and Glucocorticoid Receptor (D6H2L) XP^® Rabbit mAb #12041, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows the specific binding of GR across SLC19A2, a known target gene of GR (upper track), compared to the input control (lower track). Forty-one percent (41%) of the GR antibody-enriched DNA fragments contain the known glucocorticoid response element binding motif GNACANNNTGTNC.

Antibody Specificity for Protein Complexes

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, followed by treatment with β-estradiol (10 nM, 45 min) and either SMARCC1/BAF155 (D7F8S) Rabbit mAb #11956, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb #21792, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. As expected, all three protein subunits of the SWI/SNF complex show overlapping binding across pS2/TFF1, a known target gene of the SWI/SNF complex.