simplify genome-wide ChIP analysis for Next-Generation sequencing


Please refer to our alternative product to construct ChIP-seq libraries for Next Generation Sequencing, the Next Gen DNA Library Kit (Cat. No. 53216).

The combination of ChIP with genome-wide analysis using Next-Generation sequencing (ChIP-Seq) is a powerful approach that can provide insights into gene regulation by identifying global binding sites for proteins of interest. Active Motif's ChIP-IT® ChIP-Seq Kit provides proven reagents, streamlined protocols and validation controls to give you confidence in successful ChIP-Seq using the Illumina® sequencing platforms. The assay utilizes the highly consistent and robust chromatin immunoprecipitation and purification procedure of the ChIP-IT High Sensitivity® Kit, includes Active Motif's ChIP-IT® qPCR Analysis Kit for ChIP DNA validation and also contains a set of library construction reagents suitable for the preparation of ten Next-Gen sequencing libraries.


ChIP-IT® ChIP-Seq Method

ChIP-Seq is a multi-step process that includes chromatin preparation, immunoprecipitation, DNA purification, ChIP DNA validation and sequencing library construction. The procedure requires multiple days to complete, therefore it is strongly advised to plan your experiments in advance. The ChIP-IT® ChIP-Seq kit provides validated reagents to perform 16 chromatin preparation and immunoprecipitation reactions, 10 standard curves for ChIP DNA validation and 10 sequencing library constructions for use with the Illumina sequencing platforms. We recommend using 30 µg chromatin (4.5 million cell equivalents) per chromatin immunoprecipitation to ensure sufficient ChIP DNA for library construction.

ChIP-IT ChIP-Seq Flow Chart


Chromatin Preparation & Immunoprecipitation

First, intact cells or fresh or frozen tissues are fixed with a specially formulated formaldehyde buffer, which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication (such as with Active Motif's EpiShear® Probe Sonicator and Cooled Sonication platform) and incubated with a ChIP-validated antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration using ChIP filtration columns. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K and the DNA is recovered and purified.


ChIP DNA validation

The ChIP-enriched DNA is then validated by qPCR to confirm the quality of the DNA prior to use in sequencing library construction. The ChIP-IT® ChIP-Seq Kit includes Active Motif's ChIP-IT® qPCR Analysis Kit to perform ChIP DNA validation. The ChIP-IT qPCR Analysis Kit uses DNA standards to create a single qPCR standard curve that can be used to determine PCR primer pair efficiency. Human and mouse positive and negative control qPCR primers are included to help with data interpretation. Following qPCR amplification, the output of the qPCR instrument can be copied into the ChIP-IT qPCR Analysis Spreadsheet, which is available on our webpage for the ChIP-IT® qPCR Analysis Kit. The spreadsheet performs the calculations to normalize the data with respect to primer pair efficiency, the amount of chromatin used in each immunoprecipitation reaction and the resuspension volume of the ChIP DNA. Recommendations for data interpretation are included to evaluate the success of the ChIP reactions based on the historical experience of Active Motif using this method to analyze hundreds of ChIP reactions. Following the suggested thresholds will provide confidence that the ChIP DNA is of high quality for use in downstream sequencing.

Example data analysis of good and poor ChIP qPCR results


Sequencing Library Construction

Following validation of the ChIP-enriched DNA, single end, paired end or barcoded libraries can be generated for use on the Illumina® Genome Analyzer, HiSeq or MiSeq platforms. The adapter sequences are not included in the kit and should be obtained from Illumina. Preparation of ChIP DNA for use in Next-Generation sequencing requires the modification of DNA ends and ligation of adapters. The adapter-modified DNA is then run on an agarose gel and the library is size selected and purified. High-fidelity PCR is used to amplify the library which is validated for quantity and purity before use in sequencing.

Sequencing Library Construction

ChIP-IT ChIP-Seq Data

Active Motif has validated the ChIP-IT ChIP-Seq method across multiple types with both histone and non-histone antibodies. Below are a few examples of sequencing data obtained from the ChIP-IT ChIP-Seq Kit.


ChIP-IT ChIP-Seq data using Histone H3K9ac antibody
Figure 1: Next Generation sequencing data for Histone H3K9ac antibody.

Histone H3 Acetyl Lys9 antibody (pAb) (Catalog No. 39917) was tested by ChIP-Seq using Active Motif's ChIP-IT ChIP-Seq Kit with 30 ug of chromatin from mouse liver. ChIP DNA was sequenced on the Illumina GA II and 25 million sequence tags were mapped to identify H3K9Ac binding across the genome. The image shows a 1.5 million base pair region on chromosome 15. H3K9Ac shows promoter localization at many genes and broader binding near the Gcat gene.

ChIP-IT ChIP-Seq data using SAP30 pAb
Figure 2: SAP30 binding sites identified by ChIP-Seq.

SAP30 antibody (pAb) (Catalog No. 39731) was tested by ChIP-Seq with chromatin from the K-562 human myelogenous leukemia cell line (4.5 million cell equivalents) and 5 µl of antibody. ChIP DNA was sequenced on the Illumina HiSeq and 30 million sequence tags were mapped to identify SAP30 binding sites. The image on the left shows SAP30 binding at transcriptional start sites across a 2.4 million bp region on chromosome 1. The image on the right is zoomed in to show SAP30 binding at two other start sites.

Contents & Storage

The ChIP-IT® ChIP-Seq Kit contains separate modules for each stage of the process. One module is for chromatin preparation and immunoprecipitation, another module contains Active Motif's ChIP-IT qPCR Analysis Kit for ChIP DNA validation and a third module includes the reagents needed for library preparation for Next-Generation sequencing. Reagents within each module may contain multiple storage temperatures. All components can be stored at -20°C prior to first use, then we recommend storing each component at the temperatures indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads once they have been thawed for use.

Reagents for Chromatin Preparation and Immunoprecipitation

  • RNase A (10 µg/µl); Store at -20°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • Blocker; Store at -20°C
  • 5 M NaCl; Store at RT
  • 100 mM PMSF; Store at -20°C
  • Protease Inhibitor Cocktail (PIC); Store at -20°C
  • Precipitation Buffer; Store at -20°C
  • Carrier; Store at -20°:C
  • TE, pH 8.0; Store at RT
  • Detergent; Store at RT
  • 10X PBS; Store at -20°C
  • Fixation Buffer; Store at 4°C
  • Stop Solution; Store at RT
  • Chromatin prep Buffer; Store at RT
  • ChIP Filtration Columns; Store at RT
  • ChIP Buffer; Store at RT
  • Wash Buffer AM1; Store at RT
  • Elution Buffer AM4; Store at RT
  • Protein G Agarose Beads; Store at 4°C
  • DNA Purification Binding Buffer; Store at RT
  • 3 M Sodium Acetate; Store at RT
  • DNA Purification Wash Buffer; Store at RT
  • DNA Purification Elution Buffer; Store at RT
  • DNA Purification Columns; Store at RT

Reagents for ChIP-IT qPCR Analysis Kit

  • DNA Standard AM1; Store at -20°C
  • DNA Standard AM2; Store at -20°C
  • DNA Standard AM3; Store at -20°C
  • Standard Curve Primer Pair (2 µM); Store at -20°C
  • Human Negative Control Primer Set 1 (2.5 µM); Store at -20°C
  • Human Positive Control Primer Set GAPDH-2 (2.5 µM); Store at -20°C
  • Mouse Negative Control Primer Set 1 (2.5 µM); Store at -20°C
  • Mouse Positive Control Primer Set Gapdh-2 (2.5 µM); Store at -20°C
  • 2 mM Tris-HCl, pH 8.0; Store at RT

Reagents for Sequencing Library Construction

  • 10X Reaction Buffer AM3; Store at -20°C
  • 2X Quick Ligation Buffer; Store at -20°C
  • 10X T4 DNA Ligase Buffer; Store at -20°C
  • DNA Polymerase I Klenow (5 U/µl); Store at -20°C
  • dNTP Mix, 10 mM each; Store at -20°C
  • Klenow Fragment (3´ -5´ exo-) (5 U/µl); Store at -20°C
  • Q5® High-Fidelity DNA Polymerase (2 U/µl); Store at -20°C
  • 5X Q5 Reaction Buffer; Store at -20°C
  • T4 DNA Ligase (400 U/µl); Store at -20°C
  • T4 DNA Polymerase (3 U/µl); Store at -20°C
  • 1 mM dATP; Store at -20°C