Protocols and Standards
IHEC Guidelines
The International Human Epigenomics Consortium (IHEC) has established an Assay Standards and Quality Control Working Group (Chaired by CEEHRC Network Leader Martin Hirst) to define the assays required for three distinct classes of reference epigenome, and to define standardized protocols and quality control metrics for each assay. The Working Group's recommendations are intended to provide a framework for the definition of reference epigenomes to be included within the International Human Epigenome Consortium. These recommendations are minimal standards based on current knowledge of the elements contributing to epigenomic regulation in human cells and the current state of epigenomic mapping technologies, and are reviewed and updated annually.
Standard Operating Procedures
As an additional resource, the specific epigenomics protocols used at the BC Cancer Agency Genome Sciences Centre are listed below. The linked documents represent the latest versions of all SOPs as of April 2017; if you require a different version of any of our SOPs, please contact us.
Sample preparation, nucleic acid extraction and QC
Transfer and Cutting of Tissue Samples
Snap Freezing Protocol For Tissue
Homogenization of Tissue using Tissue Lyser LT
Harvesting Cell Lines for DNA/RNA Extraction and ChIP
DNA/RNA Extraction with AllPrep (DNA) and mirVana (total RNA with small RNA) Isolation Kits
Quantifying DNA samples using the Qubit Fluorometer
Automated DNA Quantification using the dsDNA Quant-iT High Sensitivity Assay Kit and VICTOR3V
Operation and Maintenance of the Agilent 2100 Bioanalyzer for DNA samples
Operation and Maintenance of the Agilent 2100 Bioanalyzer for RNA Samples
Operation and maintenance of the Caliper LabChip GX for DNA Samples using the High Sensitivity Assay
Operation and Maintenance of the LabChipGX for RNA samples using the HT RNA Assay
Whole genome sequencing (WGS)
DNA Sonication using Sonic Dismembrator 550
Operation of the Covaris LE220
96-well PCR free Library Construction for Illumina Sequencing
96-well PCR Free Library Construction on NIMBUS for Illumina Sequencing
96-well PCR-enriched Library Construction for Illumina Sequencing
Nimbus-assisted 96-well PCR-enriched Library Construction for Illumina Sequencing
96-well Genomic ~350bp-450bp insert Illumina Library Construction
Whole genome bisulfite sequencing (WGBS)
Manual Bisulfite Library Construction for Illumina Sequencing
96-well Bisulfite Library Construction for Illumina Sequencing
Bisulfite Library Construction on Hamilton NIMBUS for Illumina Sequencing
RNA-Seq
Magnetic bead-based mRNA isolation
Plate-based rRNA depletion Version 1
Plate-Based rRNA Depletion (RBD2.0)
Purification of polyA+ mRNA and mRNA(-) Flow-Through Total RNA using MultiMACS 96 Separation Unit
Strand Specific 96 Well cDNA Synthesis
96-well Plate-based Strand-specific cDNA Synthesis using Maxima H Minus
96-well Plate-based Strand-specific cDNA Synthesis using Maxima H Minus on Hamilton NIMBUS
Strand Specific 96-well Library Construction for Illumina Sequencing
MicroRNA-Seq
miRNA3 - Plate Format miRNA Library Construction
Automated MicroRNA Library Construction using randomized adapters for Illumina Sequencing
Chromatin immunoprecipitation sequencing (ChIP-Seq)
Crosslinking of Frozen or Fresh Tissue
Crosslinking Adherent Cell Lines
Crosslinking of Frozen Cell Pellet
Chromatin Immunoprecipitation (ChIP)
Native ChIP Using 100,000 Cells
Validation and QC of ChIP-Seq reagents
Validation Of Antibodies For ChIP
MODified Histone Peptide Array
Histone Modification Primer QC
Total Lysate Prep and BCA Protein Assay
Other assays
Antibody Validation Data
Antibodies used for ChIP-Seq at the BC Cancer Agency Genome Sciences Centre are validated using the MODifiedTM Histone Peptide Array from Active Motif; ChIP and subsequent qPCR on genomic regions annotated as enriched or depleted for the histone modification in question; and Western blotting using whole cell extracts to test non-histone affinities.SOPs for all validation protocols are available above.
Validation data for each antibody lot used at our centre are available here (PDF) - last update April 10, 2017. Information about the antibodies used to generate specific ChIP-Seq libraries is available in the corresponding metadata files.
Data Analysis Methods
Index of all data analysis methods used.
