January 2023

ChIP-seq Service Guidelines
In order to optimize results, we adhere to stringent guidelines for sample preparation (cellcollection and fixation). Please ask your Diagenode contact for your project specific guidelines.
Standard quantities per IP:- ChIP on transcription factors: >4 million cells- ChIP on histones: >1 million cells- ChIP on low cell amount (histone only): >20,000-500,000 cells
Your Service specialist will help you to determine the cell amount per sample needed for yourproject.

ChIP-seq workflow
1. Sample/Ab validation service
Use of a representative sample (each cell/tissue type).
1.1. Chromatin shearing validation• Test of two shearing times in duplicate using the Bioruptor®.
• DNA analysis to assess the shearing profile.
1.2. ChIP validation
Test ChIP (for each target) with 2 antibody quantities using iDeal ChIP-seq Kits (standardamounts) or True MicroChIP kit (low input).
By qPCRIf available, primers against control regions (one positive and one negative) should be
provided by the customer. Otherwise Diagenode recommends primer design based on datafrom public databases or publications provided by the customer.
• Control IP for H3K4me3/PolII and IgG are included in parallel.
• Validation of qPCR primers on a serial dilution of gDNA.
• Measure of the percentage of input by qPCR, calculation of the ratio of specificenrichment.
or
By sequencing• Quantification and library preparation with the MicroPlex Kit• Illumina sequencing, Paired-reads 2x50 bp.
• Quality check, alignment to reference genome, identification of enriched regions (peakcalling)2. End-to-end ChIP-seq service
Two biological replicates are required for samples of interest.
2.1. Chromatin shearing• Utilizes optimal shearing conditions (with the Bioruptor®) based on previous validation
experiment (see above).
• DNA analysis to assess the shearing profile.
2.2. ChIP• Utilizes optimal antibody amount defined during validation experiment or based on
previous experience.
• Control IP for H3K4me3/PolII and IgG are included in parallel.
• Evaluation of the ChIP efficiency in qPCR using primers for the control regions (providedby customer or designed based on validation sequencing results).
2.3. Library preparation
Use of the MicroPlex Kit (starting material down to 500 pg of DNA).
• Control ChIP DNA is included in parallel.
• Libraries are quantified and analysed to assess their size.
2.4. Sequencing
Illumina technology, Paired-reads 2x50 bp.
Other sequencing parameter are available on request.
Marks
Input
Non-histone protein
HistoneHistoneMark category

Raw read numbers (millions)for human, mouse and rat
Input
30/50 (as respective IP)
TF
30
Narrow (H3K4me3,…)30
Broad (H3K27me3,…) / Ubiquitous 50(H3PanAc,..)2.5. Bioinformatic analysis
Quality check, alignment to reference genome, identification of enriched regions (peakcalling).
Provided files:o Report with sequencing statisticso Raw data in FASTQ format
o FastQC reportso Alignment files in BAM format
o Peak files in BED format

2.6. Additional bioinformatics analysis on request• Differential binding analysis: Identification and annotation (human, mouse, rat,drosophila) of differential binding between samples based on previously identified ChIPseq peaks.
Provided files:o Report with summary of differential binding analysis and plots,o Files containing differentially bound sites or unique peaks and breakdown of those inannotated regions: introns, exons, promoters, 1-to-5 kb upstream-TSS and intergenic
regions for human, mouse, rat and drosophila.
•Annotation in genomic regions: Annotation of ChIP-seq peaks with genomic regions:introns, exons, promoters, 1-to-5 kb upstream-TSS and intergenic regions for human,mouse, rat and drosophila.

•Gene ontology terms analysis: Enrichment analysis on gene sets. Gene Ontology terms
that are overrepresented in bound regions or differentially bound regions may indicate
the underlying biological processes involved.

•Pathway analysis: Identify biochemical pathways in which genes associated with boundregions or differentially bound regions may be overrepresented.

•Visualization of specific genomic regions: Visualization of results (i.e. sequencing data,peaks) at specific genomic regions (e.g. genes, promoters) in publication-ready images(human, mouse, rat).

2.7. Additional information
For sample preparation and sample shipment it is mandatory to follow Diagenode’s
guidelines. If customer samples do not meet Diagenode’s quality requirements, any additional
QC of new samples will be charged to the customer. Any delay in sample shipment toDiagenode’s facilities might result in delaying customer’s project.
Generated files will be available for download during 1 month and stored for an additional
period of 3 months on Diagenode’s servers. Additional long-term storage of data is available
upon request. This offer includes a one-hour call to walk you through the results if needed.
Original samples are stored at Diagenode during 4 months after project completion, but will
be discarded once this time is exceeded. Return shipment of samples is available uponrequest.
Any additional service which is beyond the current project scope will be charged to thecustomer.