MeDiP Sequencing (5-Methylcytosine (5-mC) MeDIP)

Types of Libraries:

1. Chip Sequencing
2. Medip Sequencing
3. RRBS (Reduced Representation Bisulfite)
4. Whole genome (or Oxy) Bisulfite Sequencing

Methylated DNA Immunoprecipitation (MeDIP) is an immunocapture technique in which an antibody specific for methylated cytosines is used to immunoprecipitate methylated genomic DNA fragments.

MeDIP uses single-stranded DNA that has been fragmented by sonication or enzymatic digestion. The addition of a monoclonal antibody specific for 5-methylcytosine in the presence of a bridging antibody enables capture of the DNA fragments containg 5-mC methylation.

The use of Protein G magnetic beads provides quick and efficient capture. Methylated fragments are then eluted from the magnetic beads providing DNA samples that are specifically enriched for 5-mC DNA methylation. This selectivity is important as most common approaches to analyze DNA methylation, such as enzymatic approaches and bisulfite conversion, are unable to distinguish between 5-mC and 5-hmC.

MeDIP Advantages

• Specific antibody only enriches 5-mC methylated DNA (ACTIVE MOTIFS)
• Includes bridging antibody to increase efficiency of DNA capture
• One-step IP reaction
• No need to pre-wash magnetic beads

Sequencing using HiSEQ 2500
Paired End 150 base Read length
40 Million Paired-end Reads
Standard Bioinformatics Included

Standard Medip Seq Bioinformatics:
Quality Control

1. Raw data QC.
2. Removal of low quality reads and trimming of Low quality bases.
3. Adaptor trimming.

Medip Seq Analysis:

1. Mapping of Filtered data to reference.
2. Methylated mapped region can be extracted as BED format.
3. Genome-wide DNA methylation profile.
4. DNA methylation levels in : (i) promoters, (ii) bidirectional promoters, (iii) downstream of a gene and (iv) gene (3′UTRs, 5′UTRs, coding exons and introns)
5. Differential Methylated regions (DMRs) between samples.
6. Gene Target/ Motif identifications.
7. Gene Ontology & Pathway Analysis