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.
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 online at www.activemotif.com/qPCRanalysis. 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.
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.
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.
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.
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.