Consultation and workflow
Each new project starts with a personal consultation between the researcher and Abhinav Jain, Ph.D., (email@example.com) to determine the most suitable assay for the project, all appropriate controls, and the choice of antibodies and oligos for quality control (QC) assessments of experimental samples. Dr. Jain will provide project-specific guidelines for sample requirements, sample preparation and submission to the EpiCore (Figure 1). Dr. Jain frequently communicates with the investigators to update the results and project progress and will also assist in data interpretation upon specific request. Researchers with projects that involve next-generation sequencing (NGS) should always consult a bioinformatician before planning the detailed project.
Chromatin Immunoprecipitation (ChIP) Services:
ChIP followed by next-generation sequencing (NGS) is the major tool for profiling epigenetic changes and the enrichment of various of chromatin and DNA binding factors throughout the genome. In addition to traditional ChIP-Seq, the EpiCore also offers histone ChIP-seq assays in a high-throughput format (HT-ChIP), which aids in simultaneous screening of samples with multiple antibodies targeting individual transcription factors and/or specific histone modifications with minimal sample-to-sample variation. See Figure 2 for ChIP-Seq with antibodies specific to histone-PTMs is used to define chromatin states (data generated at EpiCore). Our comprehensive ChIP services encompass all necessary steps, starting from cells or tissues (details listed below):
- Chromatin Preparation: We begin by preparing high-quality chromatin from frozen pellets of fixed cells or flash-frozen tissues. To ensure chromatin integrity, we perform quality control measures before proceeding with the ChIP assay. For specific sample preparation guidelines, please contact us.
- Protein-bound DNA Pull-down (ChIP) using either antibodies recognizing specific histone post-translational modifications (PTMs), available at EpiCore (listed below) or user preferred custom antibodies*. To assess the quality of the ChIP assay, appropriate negative and positive control antibodies are also included.
- ChIP-qPCR: We offer the option to detect the enrichment of the protein of interest on a specific genomic locus through ChIP-qPCR.
- Library Preparation for ChIP-Seq: To investigate the enrichment of the protein of interest genome-wide, we prepare libraries for NGS (ChIP-Seq). After undergoing quality control at the EpiCore, these libraries are submitted to your preferred NGS facility for sequencing.
- Quality Control (QC): Throughout the entire process, both the ChIP DNA and libraries undergo quality control at multiple steps to ensure data accuracy and reliability.
With this comprehensive workflow, our ChIP services at the EpiCore are geared towards providing valuable insights into chromatin dynamics and epigenetic regulation, leading to a deeper understanding of gene expression control and broader genomic processes.
The following histone PTM antibodies are available through the EpiCore. These antibodies target specific histone modifications associated with distinct genomic regions, helping to elucidate chromatin states and gene regulatory elements.
- H3K4me3, H3K9ac and H3K27me3: These antibodies target histone H3 trimethylated at lysine 4 (H3K4me3), histone H3 acetylated at lysine 9 (H3K9ac) and histone H3 trimethylated at lysine 27 (H3K27me3). They are associated with promoters and play a crucial role in regulating gene expression.
- H3K4me1 and H3K27ac: These antibodies recognize histone H3 monomethylated at lysine 4 (H3K4me1) and histone H3 acetylated at lysine 27 (H3K27ac). They are indicative of enhancers, regions that enhance the activity of nearby genes.
- H3K36me3 and H3K79me2: These antibodies target histone H3 trimethylated at lysine 36 (H3K36me3) and histone H3 dimethylated at lysine 79 (H3K79me2). They are associated with gene bodies and active transcription.
- H3K9me3: This antibody is specific to histone H3 trimethylated at lysine 9 (H3K9me3) and is associated with heterochromatin, regions of condensed chromatin with low transcriptional activity.
*Investigator-preferred antibodies can also be used. These must be provided by the customer and should have been previously validated for ChIP. The EpiCore has successfully performed ChIP-Seq with more than 50 investigator-supplied, non-histone antibodies.
CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is a revolutionary, cost-effective, technique for determining chromatin occupancy of a specific protein with fewer cells (~500,000 in most cases). CUT&RUN requires fewer sequencing reads than traditional ChIP-seq methods, eliminates the need for a cross-linking step, and overcomes the problem of fragmentation bias.
In CUT&RUN, the protein of interest while bound to chromatin is targeted by an antibody specific for that protein. The antibody is then targeted by a recombinant protein A-micrococcal nuclease fusion that cuts and releases DNA fragments from bulk chromatin at the sites of antibody binding. The EpiCore offers the following comprehensive CUT&RUN services, starting from cells (details listred below). See Figure 3 for CUT&RUN specific to histone-PTM antibodies to define chromatin states (data generated at EpiCore). Our CUT&RUN services include the following steps:
- Harvesting and Nuclei Isolation: We harvest the required number of cells and isolate nuclei, as required.
- Protein-bound DNA Pull-down using either antibodies recognizing specific histone post-translational modifications (PTMs), available at EpiCore (listed above) or user preferred custom antibodies*. To assess the quality of the CUT&RUN assay, appropriate negative and positive control antibodies are included.
- Library Preparation for Next-generation Sequencing: To investigate the enrichment of the protein of interest genome-wide, we prepare libraries for NGS. After undergoing quality control at the EpiCore, these libraries are submitted to your preferred NGS facility for sequencing.
- Quality Control (QC): We perform assessments at discrete points throughout the process for QC
*Investigator-preferred antibodies can be used. These must be provided by the customer and should have been previously validated. Please discuss antibody and control requirements with Dr. Jain.
The EpiCore offers full service ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing) services. ATAC-seq is a powerful tool that aids in addressing scientific questions regarding chromatin accessibility, transcription factor binding, and nucleosome positioning across the genome. The ATAC-seq services offered at the EpiCore can be performed with fewer cells than MNase-seq, DNase-seq and FAIRE-seq typically require. Our services include all of the processing and quality control (QC) steps listed below, starting from cells/tissues.
- Harvesting and Nuclei Isolation: We harvest the required number of cells and isolate nuclei to ensure optimal starting material for the ATAC-seq assay.
- Transposition Reaction: We perform the transposition reaction, a crucial step in preparing open chromatin for subsequent analysis.
- DNA Library Preparation: We NGS ready libraries from the open chromatin to profile chromatin accessibility, enabling researchers to gain insights into various genomic elements.
- Assessment of Open Chromatin by qPCR: To validate the results and ensure accuracy, we assess the enrichment of open chromatin by qPCR at validated genomic locations, using primers pre-designed by EpiCore.
- Quality Control (QC): At discrete points throughout the ATAC-seq process, we perform quality control assessments to maintain the reliability and consistency of the data.