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

Purification of polyA+ mRNA and mRNA(-) Flow-Through Total RNA using Nimbus-assisted 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)

qPCR of ChIP Samples

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

Western Blot

Other assays

Tethered Conformation Capture

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.