MagMeDIP Kit
Magnetic Methylated DNA
Immunoprecipitation Kit
Cat. No. C02010020 (10 rxns)
C02010021 (48 rxns)

USER GUIDE
Version 5 05_2023

Contents
Introduction ........................................................................................................................................ 5
Kit Method Overview ......................................................................................................................... 7
Kit Materials ........................................................................................................................................ 8
Required Materials Not Provided..................................................................................................... 9
Remarks Before Starting ................................................................................................................. 11
Manual Processing ........................................................................................................................... 15
Automated Processing ................................................................................................................28
Example of Results ........................................................................................................................... 47
FAQs................................................................................................................................................... 51
Related Products .............................................................................................................................. 54
Revision history ................................................................................................................................ 54

The MagMeDIP Kit has been validated on the IP-Star®
Compact Automated System. Two versions of this protocol(manual and automated) are described in this document.

Please read this manual carefully
before starting your experiment

4

MAGMEDIP KIT MANUALIntroduction
DNA methylation is a key epigenetic mechanism with important regulatory functions inbiological processes such as genomic imprinting, control of transcription, embryonicdevelopment, X-chromosome inactivation, chromosome stability, and carcinogenesis.
DNA methylation occurs primarily as 5-methylcytosine (5-mC), and the Diagenode MagMeDIP
Kit takes advantage of a specific antibody targeting this 5-mC to immunoprecipitatedmethylated DNA, which can be thereafter directly analyzed by qPCR or Next-Generation
Sequencing (NGS). This methylation analysis is fast and highly specific as theimmunoprecipitation (IP) assay has been optimized to specifically select and precipitate themethylated DNA by the use of our 5-mC monoclonal antibody 33D3 (Cat. No. C15200081),
optimized buffers and protocol. This kit is provided with a manual protocol as well as an
automated version on Diagenode IP-Star Compact Automated System.
The MagMeDIP Kits are available in two formats for 10 and for 48 immunoprecipitation
reactions (IPs).
The kit includes:• A XL GenDNA Extraction Module to prepare large amounts of DNA from cultured
cells• Protocol for DNA shearing using Diagenode's Bioruptor• All the reagents for the IP, including 5-mC antibody 33D3 and magnetic beads• Methylated and unmethylated DNA spike-in controls and their associated qPCR
primers, allowing quality control of the IP• Reagents for fast DNA isolation after IP for qPCR application
We recommend Diagenode’s magnetic rack DiaMag 0.2ml (Cat. No. B04000001) together
with our MagMeDIP Kits. Diagenode’s magnetic racks are designed to be used in IP
experiments, keeping samples cool longer and allowing the use of small tubes to reduce thereaction volumes and use of reagents.
The kit ensures the use of a low amount of antibodies and buffers per reaction. The numberof steps is reduced and handling is easier which makes this kit cost effective and simple towork with.
The MagMeDIP Kit can be used with DNA from every species.

5Diagenode's MagMeDIP Kits have been cited in many publications and chosen by leading
laboratories worldwide thanks to their numerous benefits:• Complete kits including DNA extraction module, antibody, spike-in controls andtheir corresponding qPCR primer pairs• Easy to use with user-friendly magnetic beads and rack
• Highly validated protocols compatible with qPCR and NGS downstream analyses• Automated protocol supplied

6

MAGMEDIP KIT MANUAL

Kit Method Overview

7

Kit Materials
The MagMeDIP Kit is available in two formats so that the kit content (Table 1 below) is sufficient toperform either 10 or 48 methylated DNA immunoprecipitations and 6 DNA extractions.
Table 1. Components of MagMeDIP Kit
Component

Qty (x10)

Qty (x48)

Storage

Magbeads (magnetic beads)

150 μl750 μl4°C
Do not freeze

Nuclease-free water

4 ml

10 ml

4°C

5x MagBuffer A **2 x 1 ml

10 ml

4°C

MagBuffer B

100 μl500 μl4°C

MagBuffer C

40 μl

200 μl-20°C

Antibody anti-5mC* (33D3 clone)

5 μl

25 μl

-80°C

Methylated spike-in control

6 μl

28 μl

-20°C

Unmethylated spike-in control

6 μl

28 μl

-20°C

MagWash buffer-1 **

6 ml

30 ml

4°C

MagWash buffer-2

4 ml

20 ml

4°C

DNA Isolation Buffer (DIB)

4 ml

20 ml

4°C

Proteinase K (100x)

20 μl

200 μl-20°C

Primer pair for Methylated spike-in ctrl (5μM each)
Primer pair for Unmethylated spike-in ctrl (5μM
each)
200 µl tube strips (8 tubes/strip)

50 μl

288 μl

-20°C

50 μl

288 μl

-20°C

2

8

RT

Cap strips

2

8

RT

GenDNA Digestion Buffer

3 ml

3 ml

4°C

GenDNA Proteinase K (200X)

300 µg / 15 µl

300 µg / 15 µl-20°C

GenDNA Precipitant

3 ml

3 ml

4°C

GenDNA TE

3 ml

3 ml

4°C

5 µg / 10 µl

5 µg / 10 µl-20°C

GenDNA RNase (DNAse free)

NOTE: Upon receipt, store the components at the indicated temperatures.
* Avoid freeze-thawing cycles of this very sensitive antibody by aliquoting it before storage at -80°C.

** The composition of two buffers (5x MagBuffer A and MagWash buffer-1) has been slightly
modified. The performance of the kit has been thoroughly tested and we confirm that this modification
does not influence the kit performance. The change applies to all kits produced from December 2022
(lot 26 and the future lots).

8

MAGMEDIP KIT MANUALRequired Materials Not Provided
Materials and Reagents• Gloves to wear at all steps• DNase-free 1.5 ml and 15 ml conical tubes• Ice-cold PBS buffer• Phenol/chloroform/isoamyl alcohol (25:24:1)• Chloroform/isoamyl alcohol• Ethanol
Equipment• Diagenode magnetic rack: DiaMag 0.2ml (B04000001)• Magnetic rack for 1.5 ml tubes• Centrifuges for 1.5 ml tubes and 15 ml conical tubes (4°C)• Microcentrifuge for 0.2 ml tubes• Thermomixer (95°C)• Thermocycler• Cell counter• Rotating wheel• Diagenode Bioruptor® sonication device and the associated tube holders andtubes:- Bioruptor® Pico (Cat No. B01080010) or- Bioruptor® Plus (Cat No. B01020001)• Reagents and equipment for quantitative PCR• Reagents and equipment for DNA analysis such as electrophoresis on agarose gel,
BioAnalyzer (Agilent) or Fragment Analyzer (Agilent)• Reagents and equipment for DNA quantification such as Qubit® Fluorometer(ThermoFisher Scientific)

9Required supplies if working with automated protocol
Component

Cat. No.

Format

IP-Star Compact Automated System

B03000002

1 unit200 μl tubes strips (8 tubes/strip) + cap strips

C30020002

120 pcTips (box)C30040021

10 x 96 tips

Tips (bulk)

C30040020

1000 pc

2 ml microtubes

C30010014

100 pcMedium reagent container

C30020003

10 pc

96 well microplates

C30080030

10 pc

Additional supplies (included and available separately)
Component

Cat. No.

FormatXL GenDNA Extraction Module

C03030020

6 rxnsAntibody anti-5-mC (33D3 clone)

C15200081

10/100/500 μg

DiaMag anti-mouse IgG coated magnetic beads

C03010022

1.5 mlDNA methylation control package V2

C02040019

48 rxns

Optional supplies (not included and available separately)
Component

Cat. No.

FormatDNA Methylation control package

C02040012

40 rxns

C17011041

50/500 μl

C17011047

50/500 μl

C17021042

50/500 μl

C17021045

50/500 μl

C17031043

50/500 μl

C17031046

50/500 μl

Human TSH2B promoter primer pair (methylated
region)Human ChIP-seq grade GAPDH TSS primer pair(unmethylated region)
Mouse TSH2B coding region primer pair (methylated
region)Mouse GAPDH promoter primer pair (unmethylated
region)Rat TSH2B coding region primer pair (methylated
region)Rat GAPDH promoter +0.3 kb primer pair(unmethylated region)

10

MAGMEDIP KIT MANUAL

Remarks Before Starting
DNA extraction
The quality of the DNA to be used in MagMeDIP is important. Thus, we recommend using
Diagenode XL GenDNA Extraction Module (included in this kit) for the DNA extraction. It was
optimized for the preparation of genomic DNA (gDNA) from cultured cells. For DNA extraction
from blood or tissue - other specific kits should be used.
The XL GenDNA Extraction Module for gDNA isolation provides sufficient volume of reagentsfor the preparation of 6 gDNA batches, each obtained from 1 to 1.5 million cultured cells.
Starting amountThe MagMeDIP Kit can be used with DNA amounts ranging from 10 ng to 1 µg of sheared DNA
per IP.
Guidelines regarding the required amount of cells:• From 1.5 million cells, 8 to 12 μg of gDNA can be expected• Some of the isolated gDNA will be used as a control for DNA preparation efficiency• At least 10 ng of sheared DNA is needed per IP. Please note that since low starting
amount of DNA leads to higher background, we recommend using as much DNA as
possible• Some of the sheared DNA will be used as a control checking for: (a) shearing
efficiency and (b) the IP experiment efficiency (see section Input below)
DNA shearing
For an efficient and best resolution IP experiment, the gDNA has to be sheared into fragments
around 400 bp for MeDIP-qPCR and 200 bp for MeDIP-sequencing. High quality shearing canbe obtained with the Bioruptor Pico (Cat. No. B01080010) or the Bioruptor Plus (Cat. No.
B01020001) following Diagenode’s protocols. Before starting with IP experiment, werecommend analyzing the size of the fragments after shearing using a dedicated device suchas the BioAnalyzer (Agilent) or the Fragment Analyzer (Agilent).
Magnetic beads
This kit includes magnetic beads (MagBeads). Please make sure the beads do not dry out
during the procedure as this may result in reduced performance. Keep the MagBeads
homogenous in suspension at all times when pipetting. Variation in the number of beads will
decrease reproducibility. Do not freeze the MagBeads.
11

Input“Input sample” corresponds to the sheared DNA that undergoes the full MeDIP procedure
without incubation with the antibody and magnetic beads (Magbeads). The input sample isused as a reference to calculate the recovery by qPCR at the end of the MeDIP. We
recommend including one input sample for each sheared DNA sample that undergoes MeDIP.
Methylated and unmethylated spike-in controls
The MagMeDIP Kit contains one methylated and one unmethylated spike-in controls that canbe added directly to DNA samples before the IP. Those two spike-in controls are nothomologous to any model species and will not interfere with the sample of interest.
To check the efficiency of the MeDIP experiment, this kit also includes the primer pairs targeting the methylated and unmethylated spike-in control regions - to calculate theirrecovery by qPCR.

NOTE: The spike-in controls and the primer pairs included in the MagMeDIP Kit areavailable separately within the DNA methylation control package V2 (Cat. No.
C02040019).

CAUTION: The spike-in controls and the primer pairs provided in the separate DNA
methylation control package (Cat. No. C02040012) are different from the ones

included in the MagMeDIP Kit. Those spike-in controls are made from A. thaliana.
Therefore, they can interfere with DNA samples derived from plant species.
Technique for DNA isolation

The MagMeDIP Kit includes a DNA Isolation Buffer (DIB) for an easy and very fast DNA
isolation after MeDIP providing DNA suitable for qPCR analysis. If DNA of higher purity isneeded (e.g. for Next-Generation Sequencing), we recommend using the IPure kit v2 (Cat.
No. C03010014). The IPure kit v2 is the only DNA purification kit that is specifically optimized
for extracting very low amounts of DNA after MeDIP.
Quantitative PCR analysis – Control regions
This kit contains two primer pairs targeting the methylated and unmethylated spike-in
controls to be used in qPCR to check the success of the immunoprecipitation in eachexperiment.
Primers for methylated (TSH2B, also known as H2BC1) and unmethylated (GAPDH) control
regions in human, mouse and rat can be purchased separately (see Optional supplies, page9). Endogenous regions likeTSH2B and GAPDH allow to check the IP success on the DNA
sample rather than on the exogenous spike-in controls. This is therefore a useful tool but it
requires a minimum amount of immunoprecipitated DNA to work properly. Consequently,
depending on the sample type, it may be difficult to obtain accurate results when
immunoprecipitation is performed on less than 100 ng of sheared genomic DNA. As cell-free

12

MAGMEDIP KIT MANUALDNA is fragmented at a size close to the amplicon size, it is also not appropriate to run thoseqPCR on endogenous regions while using such template.
For each primer pair, please run the input sample alongside the IP samples. As downstream
analysis, qPCR reactions are recommended at least in duplicates (although triplicates areadvised as for potential outliers’ identification).
Quantitative PCR analysis – Regions of interest
To study DNA methylation level of a few loci of interest, MagMeDIP can be coupled with qPCR.
The primer design is thus an important step of the procedure. Here are some guidelines andtools to design good primers:• The length should be around 20 nucleotides.
• Self-complementarity and secondary structure of the primers can be tested during
primer design (https://bioinfo.ut.ee/primer3/).
• Annealing temperature of 60°C (+/- 3.0ºC) is recommended.
• Short length of amplified DNA fragment (50 – 100 bp) facilitates the PCR efficiency andreduces potential problems in amplification of G/C-rich regions.
• Difference in melting temperature between forward and reverse primers should notexceed 2 to 3°C.
• G/C stretches at the 3’ end of the primers should be avoided.
• A % GC of 50% (+/- 4%) is recommended.
After reception of candidate primer pairs a validation step is needed:• Test primer sets by in silico PCR (http://genome.cse.ucsc.edu/cgi-bin/hgPcr). Primers
should amplify unique DNA products from the genome.
• Test every primer set in qPCR using 10-fold serial dilutions of input DNA. Calculate
amplification efficiency (AE) of primer set using the following formula: AE= 10^(-1 /slope). The ideal amplification factor is 2. If it is not the case the qPCR reagents fromdifferent brand or new primers should be tested.
MagMeDIP-sequencing (MagMeDIP-seq)
For DNA methylation analysis on the whole genome, MagMeDIP can be coupled with NextGeneration Sequencing. To perform MeDIP-sequencing we recommend the following
strategy:• Choose a library preparation solution which is compatible with the starting amountof DNA you are planning to use (from 10 ng to 1 µg). It can be a home-made solution
or a commercial one.
• Choose the indexing system that fits your needs considering the following features:- Single-indexing, combinatorial dual-indexing or unique dual-indexing- Number of barcodes- Full-length adaptors containing the barcodes or barcoding at the final
amplification step13

- Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)• Standard library preparation protocols are compatible with double-stranded DNA
only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor
ligation and clean-up) will have to be performed on sheared DNA, before the IP.
CAUTION: As the immunoprecipitation step occurs at the middle of the library
preparation workflow, single-tube solutions for library preparation are usually notcompatible with MeDIP-sequencing.
• For DNA isolation after the IP, we recommend using the IPure kit v2 (available
separately, Cat. No. C03010014) instead of DNA isolation Buffer.
• Perform library amplification after the DNA isolation following the standard protocolof the chosen library preparation solution.

14

MAGMEDIP KIT MANUALPROTOCOLSTEP 1 – Cell collection & lysis

16

STEP 2 – Nucleic acid extraction & purification

17

STEP 3 – DNA shearing

19

STEP 4 – Methylated DNA immunoprecipitation

21

STEP 5 – Methylated DNA isolation

26

STEP 6 – qPCR analysis

27

MANUAL PROCESSING
15

1. STEP 1

Cell collection & lysis
Day 1

30 minutes, overnight

Starting material: cultured cells

NOTE: For recommendations about starting amounts of material, please refer tosection “Remarks before starting”.
1.1

Pellet suspension culture out of its serum containing medium or trypsinize adherent
cells and collect cells from the flask. Centrifuge at 300g for 5 minutes at 4°C.

1.2

Discard the supernatant. Resuspend cells in 5 to 10 ml ice-cold PBS. Count cells.
Centrifuge at 300 x g for 5 minutes. Discard the supernatant. Repeat this resuspension
and centrifugation step once more. This step is to wash the cells.
• Meanwhile, place the GenDNA Digestion Buffer at room temperature and the
GenDNA Proteinase K on ice (to be used at Point 1.3 below).

NOTE: If needed, cell pellets can be frozen at this step and stored at -80°C for several
months.

1.3

Prepare the complete Digestion Buffer by adding 5 μl GenDNA Proteinase K to 1 ml
GenDNA Digestion Buffer.

1.4

Resuspend cells in complete Digestion Buffer.
• For 1 to 1.5 million cells, use up to 500 μl complete Digestion Buffer• It might be necessary to use more buffer to avoid viscosity when performing theextractions

1.5

16

Incubate the samples with shaking (500 rpm) at 50°C for 12 to 18 hours in tightly
capped tubes.

MAGMEDIP KIT MANUAL

Nucleic acid extraction & purification
Day 2
1.1

PROTOCOL

STEP 2
5 hours

Thoroughly extract the samples with an equal volume of phenol/chloroform/isoamyl
alcohol (work under a fume-hood).
• Add 1 volume (500 μl according to step 1.4) of phenol/chloroform/isoamyl alcohol(25:24:1).
• Incubate the samples at room temperature for 10 minutes on a rotating wheel. Use
gentle rotation, do not vortex.

1.2

Centrifuge at 1700 x g 10 minutes.
• If the phases do not resolve properly, add another volume of GenDNA Digestion
Buffer omitting Proteinase K, and repeat the centrifugation.
• If there is a thick layer of white material at the interface between the phases, repeat
the extraction.

1.3

Transfer the aqueous (top) layer to a new tube.
• Increase volume to avoid viscosity if necessary and pipette slowly.

1.4

Thoroughly extract the samples with 500 μl of chloroform/isoamyl alcohol (onevolume).

1.5

Incubate for 10 minutes at room temperature.

1.6

Centrifuge at 1700 x g for 10 minutes.

1.7

Transfer the aqueous layer to a new 2 ml tube.

1.8

Add 250 μl of GenDNA precipitant (which is ½ volume) and 1 ml of 100% ethanol (2volumes).

1.9

Recover gDNA by centrifugation at 1700 x g for 5 minutes.
• Do not use higher speed to avoid genomic DNA fragmentation.

17• This brief precipitation in the presence of an optimized high salt precipitant(GenDNA precipitant) reduces the amount of RNA in the DNA sample. For long-term
storage, it is convenient to leave the DNA in the presence of ethanol.
1.10

Rinse the pellet with 70% ethanol. Decant ethanol and air-dry the pellet.
• It is important to rinse extensively to remove any residual salt or phenol.

1.11

Resuspend the pellet of gDNA at ~1 mg/ml in GenDNA TE until dissolved. Shake gently
at room temperature or at 65°C for several hours to facilitate solubilization. Store at
4°C.
• From 1-1.5 million cells, ~8 to 12 μg of gDNA can be expected.
• From 3 million cells, ~20 to 30 μg of gDNA can be expected.

NOTE: At this step the DNA quality can be analyzed by electrophoresis on 0.8% agarose
gel or with a Fragment Analyzer (Agilent) and Standard Sensitivity Genomic DNA
Analysis Kit (DNF-487, Agilent) (please refer to manufacturer’s instructions).

1.12

If present, residual RNA has to be removed at this step by adding 2 μl of GenDNA
RNAse (DNAse-free) per ml of DNA sample and incubating 1 hour at 37°C, followed byphenol/chloroform extraction and ethanol precipitation (same protocol as above,
starting from step 2.1).

1.13

Store the gDNA at 4°C until the shearing.

NOTE: For long term storage the gDNA can be placed at -20°C.

18

MAGMEDIP KIT MANUALDNA shearing
Day 3

PROTOCOL

2. STEP 3
2 hours

Genomic DNA must be randomly sheared by sonication on Diagenode’s Bioruptor to generate
fragments around 400 bp for qPCR application or 200bp for NGS application (see Figure 1 in
"Example of results" section).
2.1

Switch on the Bioruptor and set the temperature of the cooler at 4°C.

2.2

Prepare the gDNA solution at concentration 2-100 ng/μl using GenDNA TE.

NOTE: The smear of a sample with a concentration below 2 ng/µl will not be detectedby most instruments for quality control.
2.3

Transfer a required volume of DNA solution to the appropriate sonication microtubes:
Type

Bioruptor
Pico

Bioruptor
Plus

Samplevolume100 µl<100 µl

100 µlMicrotubes

Tube holder

0.65 ml Microtubes for
Bioruptor PicoCat. No. C30010011
0.2 ml Microtubes for
Bioruptor PicoCat. No. C30010020
0.5 ml Bioruptor Plus
Microtubes (Cat. No.
C30010013)

Tube holder for 0.65 ml
tubes - Bioruptor® PicoCat. No. B01201143
Tube holder for 0.2 ml
tubes - Bioruptor® PicoCat. No. B01201144
0.5/0.65 ml tube holder
for Bioruptor® Standard &PlusCat. No. B01200043

2.4

Briefly vortex (5-10 sec) and spin samples.

2.5

Incubate on ice for 10 minutes.

19

2.6

Shear gDNA by sonication using the Bioruptor. Choose the protocol which is adapted
to your sonicator model, sample volume and target size as described in the below
table:
Application

MagMeDIP-qPCR

MagMeDIP-seq

Target size

Bioruptor
model

SamplevolumeTubes

Shearing
protocol300-500 bp

Bioruptor
Pico

100 µl

0.65 ml Microtubes
for Bioruptor Pico

8 cycles15 sec ON
90 sec OFF

300-500 bp

Bioruptor
Pico

50 µl

0.2 ml Microtubes for
Bioruptor Pico

5 cycles
30 sec ON
30 sec OFF

300-500 bp

Bioruptor
Plus

100 µl

0.5 ml Bioruptor Plus
Microtubes

6 cycles
30 sec ON
90 sec OFF

200 bp

Bioruptor
Pico

100 µl

0.65 ml Microtubes
for Bioruptor Pico

13 cycles
30 sec ON
30 sec OFF

200 bp

Bioruptor
Pico

50 µl

0.65 ml Microtubes
for Bioruptor Pico

13 cycles
30 sec ON
30 sec OFF

200 bp

Bioruptor
Plus

100 µl

0.5 ml Bioruptor Plus
Microtubes

30 cycles
30 sec ON
90 sec OFF

CAUTION: Only use the recommended tubes for high quality results.
NOTE: To guarantee homogeneity of shearing, always completely fill the tube holder
with tubes. Never leave empty spaces in the tube holder. Fill the empty spaces withtubes containing the same volume of distilled water.

.

20

MAGMEDIP KIT MANUALMethylated DNA immunoprecipitation
Day 3-4

PROTOCOL

3. STEP 4

45 minutes, overnight

BEADS PREPARATION
Note: In order to save time, the beads preparation can also be performed during the 15
minutes incubation time of step 4.11.
3.1

Determine the number of IP reactions to be run. It is recommended to perform two
IPs per DNA sample. Input will not undergo immunoprecipitation and is therefore notconsidered as an IP.

3.2

Prepare 1x MagBuffer A (bead wash buffer) as described in the table below and keep
it on ice. The volumes contain a sufficient excess.

3.3

1 IP

2 IPs

3 IPs

4 IPs

5 IPs

5x MagBuffer A

20 μl

40 μl

60 μl

80 μl

100 μl 120 μl 140 μl 160 μl

Nuclease-free Water

80 μl

160 μl 240 μl 320 μl 400 μl 480 μl 560 μl 640 μl

9 IPs

10 IPs11 IPs

12 IPs13 IPs

6 IPs

14 IPs

7 IPs

15 IPs

8 IPs

16 IPs

5x MagBuffer A

180 μl 200 μl 220 μl 240 μl 260 μl 280 μl 300 μl 320 μl

Nuclease-free Water

720 μl 800 μl 880 μl 960 μl 1040 μl 1120 μl 1200 μl 1280 μl

Take the required amount of Magnetic beads (Magbeads) and transfer it to a clean
1.5 ml tube. 11 μl of beads are needed per IP.

NOTE: Keep beads in liquid suspension at all handling steps, as drying will result in
reduced performance. Do not vortex.

3.4

Place the tube on a magnetic rack for 1.5 ml tubes to discard the supernatant. Keep
the beads.

3.5

Wash the magnetic beads (Magbeads) twice with ice-cold 1x MagBuffer A (bead wash
buffer) as follows:21• Take the required amount of ice-cold 1x MagBuffer A and transfer it to the tube
containing the beads. 27.5 μl of 1x MagBuffer A are needed per IP• Resuspend the beads by pipetting up and down several times.
• Briefly spin the tube, place it on the magnetic rack and wait until the beads arecompletely bound to the magnet (~1 minute).
• Carefully remove and discard the supernatant without disturbing the beads.
3.6

After washing, resuspend the beads in 22 μl of 1x MagBuffer A per IP. Keep on ice.

CAUTION: Do not freeze the beads.

DNA IMMUNOPRECIPITATION
3.7

Prepare your DNA samples in a total volume of 57 µl in a 1.5 ml nuclease-free tube as
described in the following table:
Component

Quantity/Volume
10 ng to 1 µg
+ 10% surplus for inputs
Up to 57 µl

DNA*
Nuclease-free water
Total Volume

57 µl* It can be sheared DNA or adapters-ligated DNA in case of NGS application
3.8

In a new 1.5 ml tube, prepare the MagMaster Mix as described in the following table.
The volumes also include all inputs needed (1 input/IP) and contain a sufficient excess.
1 IP

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

5x MagBuffer A

24 μl

55 μl

83 μl

110 μl138 μl

166 μl193 μl221 μlMagBuffer B

6.0 μl13.8 μl20.7 μl27.6 μl34.5 μl41.4 μl48.3 μl55.2 μl

0.5 μl1.2 μl

1.7 μl2.3 μl

2.9 μl

3.5 μl4.0 μl

4.6 μl

0.5 μl1.2 μl

1.7 μl2.3 μl

2.9 μl

3.5 μl4.0 μl

4.6 μl

2.0 μl

4.6 μl

6.9 μl9.2 μl11.5 μl13.8 μl

16.1 μl18.4 μl

Methylated spike-in
control
Unmethylated spike-in
control
Nuclease-free water

22

2 IPs

9 IPs

10 IPs 11 IPs 12 IPs 13 IPs 14 IPs 15 IPs 16 IPs

5x MagBuffer A

248 μl276 μl304 μl331 μl359 μl386 μl414 μl442 μlMagBuffer B

62.1 μl69 μl75.9 μl82.8 μl89.7 μl96.6 μl103.5 μl110.4 μl

Methylated spike-in control

5.2 μl5.8 μl

6.3 μl

6.9 μl7.5 μl8.1 μl

8.6 μl9.2 μlUnmethylated spike-in
control

5.2 μl5.8 μl

6.3 μl

6.9 μl7.5 μl8.1 μl

8.6 μl9.2 μlNuclease-free water

20.7 μl23.0 μl25.3 μl27.6 μl29.9 μl32.2 μl34.5 μl36.8 μl

MAGMEDIP KIT MANUAL

Add 33 µl of MagMaster Mix to each DNA sample to form the Incubation Mix.

3.10

Incubate the Incubation Mix at 95°C for 3 minutes.

3.11

Quickly chill the Incubation Mix on ice. Perform a pulse spin to consolidate yoursample and let it cool on ice for 15 minutes.

CAUTION: Be careful to maintain the denatured DNA sample on ice until the DNA

PROTOCOL

3.9

immunoprecipitation in order to keep the DNA single-stranded and avoid re-annealing
to double-stranded DNA.
3.12

Take out 7.5 μl (that will be your 10% input) from each tube and transfer to a newlabeled 0.2 ml tube (or 8 tube-strip). Keep the input sample at 4°C: it is to be used asa control of starting material and it is therefore not to be used in IP.

3.13

Then, transfer 75 μl of Incubation Mix for each IP into one 200 μl tube (using theprovided 200 μl tube strips or individual 200 μl tubes that fit in the DiaMag 0.2ml).
Keep at 4°C.

3.14

In a new tube, dilute the antibody 1:1 with nuclease-free water as described in thefollowing table and keep on ice:

Reagent (µl)

1 IP

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

Antibody anti-5mC (33D3 clone)*

0.5 µl 0.5 µl 1.0 µl 1.0 µl 1.0 µl 1.2 µl 1.4 µl 1.6 µl

Nuclease-free water

0.5 µl 0.5 µl 1.0 µl 1.0 µl 1.0 µl 1.2 µl 1.4 µl 1.6 µl

Reagent (µl)

9 IPsAntibody anti-5mC (33D3 clone)*

1.8 µl 2.0 µl 2.2 µl 2.4 µl 2.6 µl 2.8 µl 3.0 µl 3.2 µl

Nuclease-free water

1.8 µl 2.0 µl 2.2 µl 2.4 µl 2.6 µl 2.8 µl 3.0 µl 3.2 µl

10 IPs 11 IPs 12 IPs 13 IPs 14 IPs 15 IPs 16 IPs*Avoid freeze-thawing cycles of this very sensitive antibody. For optimal storage conditions
freeze at -20°C or -80°C in small aliquots.
3.15

Prepare the Diluted Antibody Mix as described in the following table. The volumes
contain a sufficient excess. Mix the Diluted Antibody, 5x MagBuffer A and water firstand add MagBuffer C at the end.
1 IP

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

Diluted Antibody

0.30 μl0.75 μl1.05 μl1.50 μl1.80 μl2.10 μl2.40 μl

3.00 μl

5x MagBuffer A

0.60 μl1.50 μl2.10 μl

3.00 μl3.60 μl4.20 μl4.80 μl

6.00 μl

Water

2.10 μl5.25 μl7.35 μl10.50 μl12.60 μl14.70 μl16.80 μl

21.00 μl

MagBuffer C

2.00 μl

5.00 μl7.00 μl

10.00 μl

12.00 μl14.00 μl

16.00 μl

20.00 μl

TOTAL volume

5.00 μl12.50 μl17.50 μl

25.00 μl

30.00 μl35.00 μl

40.00 μl

50.00 μl23

9 IPs

10 IPs11 IPs

12 IPs13 IPs14 IPs15 IPs16 IPsDiluted Antibody

3.30 μl3.60 μl3.90 μl4.20 μl4.50 μl4.80 μl5.40 μl

6.00 μl

MagBuffer A 5x6.60 μl7.20 μl7.80 μl8.40 μl9.00 μl9.60 μl10.80 μl

12.00 μl

Water

23.10 μl25.20 μl27.30 μl29.40 μl31.50 μl33.60 μl37.80 μl

42.00 μl

MagBuffer C

22.00 μl

24.00 μl

26.00 μl

28.00 μl

30.00 μl

32.00 μl

36.00 μl

40.00 μl

TOTAL volume

55.00 μl60.00 μl

65.00 μl70.00 μl75.00 μl80.00 μl

90.00 μl

100.0 μl

3.16

Add 5 μl of Diluted Antibody Mix to each 200 μl IP tube containing the Incubation Mix(from point 4.13)

3.17

Mix and add 20 μl of washed Magbeads from point 4.6 to each 200 μl IP tube (frompoint 4.16). The final volume is 100 μl.

NOTE: Keep the beads homogenously in suspension at all times when pipetting.
Variation in the number of beads will lead to lower reproducibility.

3.18

Place the IP tube on a rotating wheel (40 rpm) at 4°C overnight.

DNA WASHES
3.19

Place the MagWash Buffer-1, MagWash Buffer-2 and the Magnetic rack – DiaMag
0.2ml on ice.

3.20

Briefly spin down the IP tube, place it on the ice-cold magnetic rack and wait until thebeads are completely bound to the magnet (~1 minute).

3.21

Carefully remove and discard the supernatant without disturbing the beads.

3.22

Wash the beads 3 times with ice-cold MagWash Buffer-1 as follows:• Add 100 µl of ice-cold MagWash Buffer-1, close the tube cap, and invert the tube
to resuspend the beads.
• Incubate at 4°C for 5 minutes on a rotating wheel (40 rpm).
• Briefly spin down the IP tube, place it on the magnetic rack and wait until the beads
are completely bound to the magnet (~1 minute).
• Carefully remove and discard the supernatant without disturbing the beads.

3.23

Wash the beads once with 100 μl ice-cold MagWash Buffer-2 as follows:• Add 100 µl of ice-cold MagWash Buffer-2, close the tube cap, and invert the tube
to resuspend the beads.
• Incubate at 4°C for 5 minutes on a rotating wheel (40 rpm).

24

MAGMEDIP KIT MANUAL• Carefully remove and discard the supernatant without disturbing the beads.
• Be sure to remove all traces of MagWash Buffer-2. If necessary, briefly spin down
the tube, place it back on the magnetic rack and remove traces of liquids with apipette.
3.24

PROTOCOL• Briefly spin down the IP tube, place it on the magnetic rack and wait until thebeads are completely bound to the magnet (~1 minute).

Place the IP tube with bead pellet on ice. The bound DNA can be now purified from
Magbeads.

25

4. STEP 5

Methylated DNA isolation
Day 4
4.1

45 minutes

Take the input samples, centrifuge briefly and from now onwards treat the input
samples and IP samples in parallel.

CAUTION: The following protocol describes the use of the DIB method to purify DNA.
This is suitable for qPCR application. For NGS application, we recommend using the
IPure Kit V2 (Cat. No. C03010014) instead of DIB.
4.2

Prepare 100 μl DNA Isolation Buffer (DIB) per sample. Add 1 μl of Proteinase K per
100 μl of DIB. Scale accordingly knowing that 100 μl are needed per IP’ed sample and92.5 μl per input sample.

4.3

Remove the tubes from the DiaMag 0.2ml and add 100 μl of DIB per IP’ed sample.
Resuspend the beads.

4.4

Add 92.5 μl of DIB to each « 7.5 μl input sample » from step 4.11.

4.5

Incubate at 55°C for 15 minutes both IP’ed samples and input samples.

4.6

Incubate all the samples at 100°C for 15 minutes.

4.7

Spin down and place the tubes into the DiaMag02.

4.8

Transfer the supernatants containing DNA in new labeled tubes, discard Magbeads.
This isolated DNA can be stored at -20°C until further processing.

NOTES:

26-

For MagMeDIP-qPCR use the isolated DNA for qPCR analyses.

-

For MagMeDIP-seq use the isolated DNA for library amplification. We
recommend performing qPCR on a small aliquot of each library for
quality control before the sequencing.

MAGMEDIP KIT MANUALqPCR analysis
Day 4

PROTOCOL

5. STEP 6> 2 hours

The MagMeDIP Kit includes two validated primer pairs:
1. Primer pair for Methylated spike-in ctrl (5μM each)
2. Primer pair for Unmethylated spike-in ctrl (5μM each)

NOTE: Primer pairs for endogenous control regions in human, mouse and rat areavailable - for more information, please visit www.diagenode.com

For large starting amounts of DNA, we advise to control the 4 DNA sequences.
For experiments starting with less than 100 ng DNA, or for MagMeDIP-seq experiments theanalysis of the two spike-in controls is enough for quality control.
5.1

Prepare the qPCR Mix for each targeted DNA sequence as described in the following
table for the number of desired reactions. Mix by pipetting and keep on ice until
further use.
Reagents

Volume for 1 reaction

Primer pair (Stock: 5 μM each)

1 μl

2x SYBR Green PCR master mix

12.5 μl

Nuclease-free water

6.5 μlTOTAL volume

20 μl

NOTES:-qPCR reagents are not provided for this step so that every researcher can use
their regular SYBRGreen Master Mix that fits best their qPCR device.
We recommend performing qPCR in duplicates or triplicates.

5.2

Transfer 20 µl of the qPCR Mix per qPCR reaction into appropriate strip or well plate.

5.3

Add 5 µl of DNA sample.

27

NOTE: The IP'ed and input samples can be diluted up to 10x in nuclease-free water
before the qPCR when needed (e.g. if libraries have to be saved for library
amplification and sequencing or if many regions have to be analyzed by qPCR).
5.4

Run the following qPCR program:
Step
1. Denaturation*
2. PCR Amplification

Temperature

Time & Cycles

95°C

7 minutes

95°C

15 seconds

60°C

60 seconds

95°C
3. Melting curve**

X 40 cycles
1 minute

Follow qPCR instrument manufacturer recommendations*Please check carefully supplier's recommendations about Taq polymerase activation
time and temperature.
**Include and inspect the melting curves to ensure that primer pairs amplify only asingle specific product.
5.5

Data analysis. Some major advices how to analyse qPCR results are given below.

IP efficiency
The efficiency of methylated DNA immunoprecipitation of a particular genomic locus can becalculated from qPCR data and reported as a recovery of starting material using the following
formula:% recovery= 2^(Ct10% input - 3.32 - CtIP sample) x 100
Here 2 is the amplification efficiency, Ct (IP sample) and Ct (10% input) are threshold values
obtained from exponential phase of qPCR for the IP sample and input sample respectively;
the compensatory factor (3.32=log2(10))) is used to take into account the 10% quantity of
the input compared to the IP quantity.

NOTE: This equation assumes that the qPCR is 100% efficient (amplification efficiency= 2). The real amplification efficiency, if known, should be used.
IP enrichment and specificity

The final goal of IP is to calculate the enrichment in the same IP sample of:• The specific DNA fragments (corresponding to the methylated DNA) in comparison
with background (corresponding to unmethylated DNA).

28

MAGMEDIP KIT MANUAL

enrichment= % recovery (specific locus) / % recovery (background locus)

IP specificity can be calculated:

PROTOCOL• This enrichment can be calculated as a ratio of specific signal over background.

Specificity = 1- (% recovery background locus/%recovery specific locus) = 1(1/enrichment)

Characteristics of a good quality IP:• Methylated spike-in recovery > 20%• Unmethylated spike-in recovery < 1%
• Spike-in IP specificity > 99%

29

STEP 1 – Cell collection & lysis

32

STEP 2 – Nucleic acid extraction & purification

33

STEP 3 – DNA shearing

35

STEP 4 – Methylated DNA immunoprecipitation

37

STEP 5 – Methylated DNA isolation

42

STEP 6 – qPCR analysis

44

AUTOMATED PROCESSING
30

MAGMEDIP KIT MANUALThe below chapter describes the protocol for MagMeDIP assay using the IP-Star Compact
Automated System (Cat. No. B03000002).

AUTO PROTOCOL

Protocol for Auto MagMeDIP using the
IP-Star Compact Automated System
If you have an older version of the IP-Star, please, contact us at
customer.support@diagenode.com for the corresponding Auto MagMeDIP manual.

31

1. STEP 1

Cell collection & lysis
Day 1

30 minutes, overnight

Starting material: cultured cells

NOTE: For recommendations about starting amounts of material, please refer tosection “Remarks before starting”.
1.1

Pellet suspension culture out of its serum containing medium or trypsinize adherent
cells and collect cells from the flask. Centrifuge at 300g for 5 minutes at 4°C.

1.2

Discard the supernatant. Resuspend cells in 5 to 10 ml ice-cold PBS. Count cells.
Centrifuge at 300 x g for 5 minutes. Discard the supernatant. Repeat this resuspension
and centrifugation step once more. This step is to wash the cells.
• Meanwhile, place the GenDNA Digestion Buffer at room temperature and the
GenDNA Proteinase K on ice (to be used at Point 1.3 below).

NOTE: If needed, cell pellets can be frozen at this step and stored at -80°C for several
months.

1.3

Prepare the complete Digestion Buffer by adding 5 μl GenDNA Proteinase K to 1 ml
GenDNA Digestion Buffer.

1.4

Resuspend cells in complete Digestion Buffer.
• For 1 to 1.5 million cells, use up to 500 μl complete Digestion Buffer• It might be necessary to use more buffer to avoid viscosity when performing theextractions

1.5

32

Incubate the samples with shaking (500 rpm) at 50°C for 12 to 18 hours in tightly
capped tubes.

MAGMEDIP KIT MANUAL

Nucleic acid extraction & purification
Day 2
2.1

AUTO PROTOCOL

2. STEP 2
5 hours

Thoroughly extract the samples with an equal volume of phenol/chloroform/isoamyl
alcohol (work under a fume-hood).
• Add 1 volume (500 μl according to step 1.4) of phenol/chloroform/isoamyl alcohol(25:24:1).
• Incubate the samples at room temperature for 10 minutes on a rotating wheel. Use
gentle rotation, do not vortex.

2.2

Centrifuge at 1700 x g 10 minutes.
• If the phases do not resolve properly, add another volume of GenDNA Digestion
Buffer omitting Proteinase K, and repeat the centrifugation.
• If there is a thick layer of white material at the interface between the phases, repeat
the extraction.

2.3

Transfer the aqueous (top) layer to a new tube.
• Increase volume to avoid viscosity if necessary and pipette slowly.

2.4

Thoroughly extract the samples with 500 μl of chloroform/isoamyl alcohol (onevolume).

2.5

Incubate for 10 minutes at room temperature.

2.6

Centrifuge at 1700 x g for 10 minutes.

2.7

Transfer the aqueous layer to a new 2 ml tube.

2.8

Add 250 μl of GenDNA precipitant (which is ½ volume) and 1 ml of 100% ethanol (2volumes).

2.9

Recover gDNA by centrifugation at 1700 x g for 5 minutes.
• Do not use higher speed to avoid genomic DNA fragmentation.
33• This brief precipitation in the presence of an optimized high salt precipitant(GenDNA precipitant) reduces the amount of RNA in the DNA sample. For long-term
storage, it is convenient to leave the DNA in the presence of ethanol.

2.10

Rinse the pellet with 70% ethanol. Decant ethanol and air-dry the pellet.
• It is important to rinse extensively to remove any residual salt or phenol.

2.11

Resuspend the pellet of gDNA at ~1 mg/ml in GenDNA TE until dissolved. Shake gently
at room temperature or at 65°C for several hours to facilitate solubilization. Store at
4°C.
• From 1-1.5 million cells, ~8 to 12 μg of gDNA can be expected.
• From 3 million cells, ~20 to 30 μg of gDNA can be expected.

NOTE: At this step the DNA quality can be analyzed by electrophoresis on 0.8% agarose
gel or with a Fragment Analyzer (Agilent) and Standard Sensitivity Genomic DNA
Analysis Kit (DNF-487, Agilent) (please refer to manufacturer’s instructions).

2.12

If present, residual RNA has to be removed at this step by adding 2 μl of GenDNA
RNAse (DNAse-free) per ml of DNA sample and incubating 1 hour at 37°C, followed byphenol/chloroform extraction and ethanol precipitation (same protocol as above,
starting from step 2.1).

2.13

Store the gDNA at 4°C until the shearing.

NOTE: For long term storage the gDNA can be placed at -20°C.

34

MAGMEDIP KIT MANUALAUTO PROTOCOL

3. STEP 3
DNA shearing
Day 3

2 hours

Genomic DNA must be randomly sheared by sonication on Diagenode’s Bioruptor to generate
fragments around 400 bp for qPCR application or 200bp for NGS application (see Figure 1 in
"Example of results" section).
3.1

Switch on the Bioruptor and set the temperature of the cooler at 4°C.

3.2

Prepare the gDNA solution at concentration 2-100 ng/μl using GenDNA TE.

NOTE: The smear of a sample with a concentration below 2 ng/µl will not be detectedby most instruments for quality control.
3.3

Transfer a required volume of DNA solution to the appropriate sonication microtubes:
Type

Samplevolume100 µlBioruptor Pico<100 µl
Bioruptor Plus

100 µlMicrotubes
0.65 ml Microtubes for
Bioruptor PicoCat. No. C30010011
0.2 ml Microtubes for
Bioruptor PicoCat. No. C30010020
0.5 ml Bioruptor Plus
Microtubes (Cat. No.
C30010013)

3.4

Briefly vortex (5-10 sec) and spin samples.

3.5

Incubate on ice for 10 minutes.

Tube holder
Tube holder for 0.65 ml tubes Bioruptor® PicoCat. No. B01201143
Tube holder for 0.2 ml tubes Bioruptor® PicoCat. No. B01201144
0.5/0.65 ml tube holder for
Bioruptor® Standard & Plus
Cat. No. B01200043

35

3.6

Shear gDNA by sonication using the Bioruptor. Choose the protocol which is adapted
to your sonicator model, sample volume and target size as described in the below
table:
Application

MagMeDIP-qPCR

MagMeDIP-seq

Target size

Bioruptor
model

SamplevolumeTubes

Shearing
protocol300-500 bp

Bioruptor
Pico

100 µl

0.65 ml Microtubes
for Bioruptor Pico

8 cycles15 sec ON
90 sec OFF

300-500 bp

Bioruptor
Pico

50 µl

0.2 ml Microtubes
for Bioruptor Pico

5 cycles
30 sec ON
30 sec OFF

300-500 bp

Bioruptor
Plus

100 µl

0.5 ml Bioruptor Plus
Microtubes

6 cycles
30 sec ON
90 sec OFF

200 bp

Bioruptor
Pico

100 µl

0.65 ml Microtubes
for Bioruptor Pico

13 cycles
30 sec ON
30 sec OFF

200 bp

Bioruptor
Pico

50 µl

0.65 ml Microtubes
for Bioruptor Pico

13 cycles
30 sec ON
30 sec OFF

200 bp

Bioruptor
Plus

100 µl

0.5 ml Bioruptor Plus
Microtubes

30 cycles
30 sec ON
90 sec OFF

CAUTION: Only use the recommended tubes for high quality results.
NOTE: To guarantee homogeneity of shearing, always completely fill the tube holder
with tubes. Never leave empty spaces in the tube holder. Fill the empty spaces withtubes containing the same volume of distilled water.

36

MAGMEDIP KIT MANUALMethylated DNA immunoprecipitation
Day 3-4

AUTO PROTOCOL

4. STEP 4

1 hour hands on time and overnight run

A – Protocol Set-up
4.1

Switch ON the IP-Star Compact.

4.2

Select “Protocols” icon and then “MeDIP” category.

4.3

Select “MeDIP_8_DIB” if you plan to run between 1 and 8 samples or“MeDIP_16_DIB” if you plan to run between 9 and 16 samples.

CAUTION: The following protocol describes the use of the DIB method to purify DNA.
This is suitable for qPCR application. For NGS application we recommend using the
IPure Kit V2 (Cat. No. C03010014) instead of DIB and the MeDIP_8_IPure or
MeDIP_16_IPure programs. For more information refer to the manual of the kit IPure
v2.

4.4

Set up the exact number of samples for your experiment by pressing the black box.
This number includes each IP and it is recommended to perform the IP in duplicates.
Input will not undergo immunoprecipitation in the IP-Star and is therefore notconsidered as a sample.

NOTE: The Peltier block is now cooling down to 4°C to keep your samples cold.
4.5

Setup the parameters for your MagMeDIP experiment and press “Next”.
37

Recommended parameters:

4.6

Setup all the plastic consumables on the platform according to the screen layout• Fill TIP Rack 1 (and 2 if processing more than 8 samples) with tips according to thescreen.
• Fill Reagent Racks 1 & 2 with Reagent containers according to the screen.

NOTE: For qPCR applications – DIB (included in the kit MagMeDIP) should be used; for
NGS applications – Elution Buffer (from the kit IPure v2) should be used.
• Fill Peltier Block 1 (and Block 2 if processing more than 8 samples) with strips according
to the screen.

NOTE: All the rows of the Peltier(s) Block(s) must be filled with a strip.

MAGMEDIP KIT MANUAL

9 10 11 12
8
7
6
2

3

4

5

96 plate 2

G

H

D
E
F

1
G

H

D
E
F

C

A

7
6

38

B

A

1

2

3

4

5

96 plate 1

C

Reagent
Rack 2

Peltier
Block 2

B

Reagent
Rack 1

Peltier
Block 1

9 10 11 12

TIP Rack 2

12
11
10
9
8
7
6
5
4
3
2
1

8

TIP Rack 1

12
11
10
9
8
7
6
5
4
3
2
1

Prepare the mixes and fill the strips with your samples and the reagents from the kit
as described below. Make sure that the liquid is at the bottom of each well.

B – Prepare Reagent and Mixes
4.8

In a new 1.5 ml tube, prepare 1x MagBuffer A (bead wash buffer) as described in thetable below. The volumes contain a sufficient excess. Keep the reagent on ice.
1 IP

4.9

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

5x MagBuffer A

50 μl

80 μl

110 μl130 μl160 μl190 μl210 μl240 μlWater

200 μl

320 μl

440 μl

520 μl640 μl760 μl840 μl960 μl

9 IPs

10 IPs11 IPs

12 IPs13 IPs14 IPs15 IPs16 IPs

5x MagBuffer A

260 μl290 μl

320 μl340 μl

370 μl400 μl420 μl450 μlWater

1040 μl

1160 μl

1280 μl

1360 μl

1480 μl

1600 μl1680 μl1800 μl

AUTO PROTOCOL

4.7

Prepare your DNA samples in a total volume of 57 µl in a 1.5 ml nuclease-free tube as
described in the following table:
Component

Quantity/Volume
10 ng to 1µg+ 10% surplus for inputs
Up to 57 µl

DNA*
Nuclease-free water
Total Volume

57 µl* It can be sheared DNA or adapters-ligated DNA for NGS application
4.10

In a new 1.5 ml tube, prepare the Mag master mix as described in the following table.
The volumes also include all inputs needed (1 input/IP) and contain a sufficient excess.
Keep the mix on ice.
1 IP

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

5x MagBuffer A

24 μl

55 μl

83 μl

110 μl138 μl

166 μl193 μl221 μlMagBuffer B

6.0 μl13.8 μl 20.7 μl 27.6 μl 34.5 μl 41.4 μl 48.3 μl 55.2 μl

0.5 μl1.2 μl

1.7 μl2.3 μl

2.9 μl

3.5 μl4.0 μl

4.6 μl

0.5 μl1.2 μl

1.7 μl2.3 μl

2.9 μl

3.5 μl4.0 μl

4.6 μl

2.0 μl

4.6 μl

6.9 μl9.2 μl11.5 μl 13.8 μl 16.1 μl 18.4 μl

Methylated spike-in
control
Unmethylated spike-in
control
Nuclease-free water39

9 IPs11 IPs

12 IPs13 IPs14 IPs15 IPs16 IPs

5x MagBuffer A

248 μl 276 μl 304 μl

331 μl359 μl386 μl414 μl442 μlMagBuffer B

62.1 μl69 μl75.9 μl82.8 μl89.7 μl 96.6 μl 103.5 μl 110.4 μl

5.2 μl5.8 μl

6.3 μl

6.9 μl7.5 μl8.1 μl

8.6 μl9.2 μl

5.2 μl5.8 μl

6.3 μl

6.9 μl7.5 μl8.1 μl

8.6 μl9.2 μl20.7 μl23.0
μl25.3 μl27.6 μl29.9 μl 32.2 μl

34.5 μl36.8 μl

Methylated spike-in
control
Unmethylated spike-in
control
Nuclease-free water

10 IPs4.11

Add 33 µl of MagMaster Mix to each DNA sample to form the Incubation Mix.

4.12

Incubate the Incubation Mix at 95ºC for 3 minutes.

4.13

Quickly chill the Incubation Mix on ice. Perform a pulse spin to consolidate yoursample and let it cool on ice for 15 minutes.

CAUTION: Be careful to maintain the denatured DNA sample on ice until the DNA
immunoprecipitation in order to keep the DNA single-stranded and avoid re-annealing
to double-stranded DNA.
4.14

Take out 7.5 μl (that will be your 10% input) from each tube and transfer to a newlabeled 0.2 ml tube (or 8tube-strip). Keep the input sample at 4°C: it is to be used asa control of starting material and it is therefore not to be used in IP.

4.15

Then, transfer 75 μl of incubation mix for each IP into one 200 μl IP-Star tube strip.
Keep at 4°C.

4.16

In a new tube, dilute the antibody 1:1 with nuclease-free water as described in thefollowing table and keep on ice:

Reagent (µl)

1 IP

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

Antibody anti-5mC (33D3 clone)*

0.5 µl 0.5 µl 1.0 µl 1.0 µl 1.0 µl 1.2 µl 1.4 µl 1.6 µl

Nuclease-free water

0.5 µl 0.5 µl 1.0 µl 1.0 µl 1.0 µl 1.2 µl 1.4 µl 1.6 µl

Reagent (µl)

9 IPsAntibody anti-5mC (33D3 clone)*

1.8 µl 2.0 µl 2.2 µl 2.4 µl 2.6 µl 2.8 µl 3.0 µl 3.2 µl

Nuclease-free water

1.8 µl 2.0 µl 2.2 µl 2.4 µl 2.6 µl 2.8 µl 3.0 µl 3.2 µl

10 IPs 11 IPs 12 IPs 13 IPs 14 IPs 15 IPs 16 IPs*Avoid freeze-thawing cycles of this very sensitive antibody. For optimal storage conditions
freeze at -20°C or -80°C in small aliquots.

40

MAGMEDIP KIT MANUALPrepare the Diluted Antibody Mix as described in the following table. The volumes
contain a sufficient excess. Mix the Diluted Antibody, 5x MagBuffer A and water firstand add MagBuffer C at the end.
1 IP

2 IPs

3 IPs

4 IPs

5 IPs

6 IPs

7 IPs

8 IPs

Diluted Antibody

0.30 μl0.75 μl1.05 μl1.50 μl1.80 μl2.10 μl2.40 μl

3.00 μl

5x MagBuffer A

0.60 μl1.50 μl2.10 μl

3.00 μl3.60 μl4.20 μl4.80 μl

6.00 μl

Water

2.10 μl5.25 μl7.35 μl10.50 μl12.60 μl14.70 μl16.80 μl

21.00 μl

MagBuffer C

2.00 μl

5.00 μl7.00 μl

10.00 μl

12.00 μl14.00 μl

16.00 μl

20.00 μl

TOTAL volume

5.00 μl12.50 μl17.50 μl

25.00 μl

30.00 μl35.00 μl

40.00 μl

50.00 μl

9 IPs

10 IPs11 IPs

12 IPs13 IPs14 IPs15 IPs16 IPsDiluted Antibody

3.30 μl3.60 μl3.90 μl4.20 μl4.50 μl4.80 μl5.40 μl

6.00 μl

5x MagBuffer A

6.60 μl7.20 μl7.80 μl8.40 μl9.00 μl9.60 μl10.80 μl

12.00 μl

Water

23.10 μl25.20 μl27.30 μl29.40 μl31.50 μl33.60 μl37.80 μl

42.00 μl

MagBuffer C

22.00 μl

24.00 μl

26.00 μl

28.00 μl

30.00 μl

32.00 μl

36.00 μl

40.00 μl

TOTAL volume

55.00 μl60.00 μl

65.00 μl70.00 μl75.00 μl80.00 μl

90.00 μl101.0 μl

AUTO PROTOCOL

4.17

C – Fill the strips with the reagents and the mixes
4.18

Distribute 10 μl of Magnetic beads (Magbeads) in each well of row 3.

NOTE: Keep the beads homogenously in suspension at all times when pipetting.
Variation in the number of beads will lead to lower reproducibility.

4.19

Prepare the Immunoprecipitation Mix in the 200 μl IP-Star tube strip from step 4.18
as described in the table below. Insert the strip in row 7.
1IP (100 μl)

MagBuffer A (1x)20 μlIncubation Mix (already in the strip)

75 μl

Antibody Mix

5 μl

NOTE: Before dispensing, mix the content in the tube by pipetting up and down.
4.20

Fill in reagent racks 1 & 2 with reagents according to the screen instructions.

4.21

Check the proper insertion of the racks and the consumables, and press “Next”.

4.22

Check the selected parameters, close the door and press “Run” to start.

4.23

MeDIP protocol is running. The “Remaining time” calculation will give you an
estimation of the processing time of your experiment.
41

5. STEP 5

Methylated DNA isolation
Day 4
5.1

10 minutes hands on time and 40 minutes run

The next morning, recover the sample tubes in row 12 (at 4°C).

NOTE: Remove all the plastics from the IP-Star.
5.2

Prepare the INPUT as described in the table below.
1 INPUT
Sheared DNA

7.5 μlDNA Isolation Buffer (DIB)*

92.5 μl*For NGS application follow the protocol for IPure kit v2 (Cat. No. C03010014).

5.3

Add 1 μl of Proteinase K in all IP samples and input samples.

5.4

Close the tubes with caps and insert them on the Peltier Block 1.

NOTE: The Peltier Block 1 heats evenly, so the strips can be placed in every row of
this block.

42

5.5

Close the door and press “OK” to start the incubation.

5.6

The incubation is now starting. The “Remaining time” calculation will give you an
estimation of the processing time.

5.7

When the protocol is completed, a screen appears telling you the run is over.
Recover the sample tubes, spin down the tubes to bring down the liquid caught in
the lid.

5.8

Place the tubes in the DiaMag 0.2ml and wait for 1 minute.

5.9

Transfer the supernatants containing DNA in new labeled tubes and discard
Magbeads. This isolated DNA is ready for qPCR analysis. Make aliquots of thisisolated DNA, store at -20°C.

MAGMEDIP KIT MANUALNow back to the IP-Star screen, press “OK” and then “Yes” to start a new run orpress “Back” until the homepage appears on the screen. Press “Shutdown” and wait
until the screen is black before switching off the IP-Star.

5.11

Remove all the plastics from the IP-Star platform, empty the waste shuttle, andclean the inner side of the IP-Star with 70% ethanol.

AUTO PROTOCOL

5.10

43

6. STEP 6
qPCR analysis
Day 4> 2 hours

The MagMeDIP Kit includes four validated primer pairs:
1. Primer pair for Methylated spike-in ctrl (5μM each)
2. Primer pair for Unmethylated spike-in ctrl (5μM each)

NOTE: Primer pairs for endogenous control regions in human, mouse and rat areavailable! For more information please visit www.diagenode.com

For large starting amounts of DNA we advise to control the 4 DNA sequences.
For experiments starting with less than 100 ng DNA, or for MagMeDIP-seq experiments theanalysis of the two spike-in controls is enough for quality control.
6.1

Prepare the qPCR Mix for each targeted DNA sequence as described in the following
table for the number of desired reactions. Mix by pipetting and keep on ice until
further use.
Reagents

Volume for 1 reaction

Primer pair (Stock: 5 μM each)

1 μl

2x SYBR Green PCR master mix

12.5 μl

Nuclease-free water

6.5 μlTOTAL volume

20 μl

NOTES:-

44

qPCR reagents are not provided for this step so that every researcher can use
their regular SYBRGreen Master Mix that fits best their qPCR device.
We recommend performing qPCR in duplicates or triplicates.

6.2

Transfer 20 µl of the qPCR Mix per qPCR reaction into appropriate strip or well plate.

6.3

Add 5 µl of DNA sample.

MAGMEDIP KIT MANUAL

before the qPCR when needed (e.g. if libraries have to be saved for library
amplification and sequencing or if many regions have to be analyzed by qPCR).
6.4

Run the following qPCR program:
Step
1. Denaturation*
2. PCR Amplification

Temperature

Time & Cycles

95°C

7 minutes

95°C

15 seconds

60°C

60 seconds

95°C
3. Melting curve**

AUTO PROTOCOLNOTE: The IP'ed and input samples can be diluted up to 10x in nuclease-free water

X 40 cycles
1 minute

Follow qPCR instrument manufacturer recommendations*Please check carefully supplier's recommendations about Taq polymerase activation
time and temperature.
**Include and inspect the melting curves to ensure that primer pairs amplify only asingle specific product.
6.5

Data analysis. Some major advices how to analyse qPCR results are given below.

IP efficiency
The efficiency of methylated DNA immunoprecipitation of a particular genomic locus can becalculated from qPCR data and reported as a recovery of starting material using the following
formula:% recovery= 2^(Ct10% input - 3.32 - CtIP sample) x 100
Here 2 is the amplification efficiency, Ct (IP sample) and Ct (10% input) are threshold values
obtained from exponential phase of qPCR for the IP sample and input sample respectively;
the compensatory factor (3.32=log2(10))) is used to take into account the 10% quantity of
the input compared to the IP quantity.

NOTE: This equation assumes that the qPCR is 100% efficient (amplification efficiency= 2). The real amplification efficiency, if known, should be used.

IP enrichment and specificity
The final goal of IP is to calculate the enrichment in the same IP sample of:• The specific DNA fragments (corresponding to the methylated DNA) in comparison
with background (corresponding to unmethylated DNA).

45

• This enrichment can be calculated as a ratio of specific signal over background.
enrichment= % recovery (specific locus) / % recovery (background locus)
IP specificity can be calculated:
Specificity = 1- (% recovery background locus/%recovery specific locus) = 1(1/enrichment)
To consider an IP to be of good quality, find below some key criteria:• Methylated spike-in recovery > 20%• Unmethylated spike-in recovery < 1%
• Spike-in IP specificity > 99%

46

MAGMEDIP KIT MANUALExample of Results
MAGMEDIP-QPCR

Figure 1. BioAnalyzer (Agilent) High Sensitivity DNA chip profile of sheared genomic DNA:smear around 400 bp.
The genomic DNA was diluted in GenDNA TE to reach a concentration of 100 ng/μl and 100
μl were sheared in a 0.65 ml Bioruptor Microtube (Cat. No. C30010011). The following
program was used: 8 Cycles: [15 seconds “ON” & 90 seconds “OFF”].

100
90
80

% Recovery

70
60
50
40
30
20
10
0

Methylated spike-in

Unmethylated spike-in
50 ng

TSH2B

GAPDH

1 µg

Figure 2. Immunoprecipitation results obtained with Diagenode MagMeDIP Kit

MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the
MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in
controls included in the kit, together with the human DNA samples. The DNA was isolated/purified
using DIB. Afterwards, qPCR was performed using the Human TSH2B promoter and GADPH TSSprimer pairs (Cat. No. C17011041 and C17011047, respectively).
47

MAGMEDIP-SEQUENCING

Figure 3. BioAnalyzer (Agilent) High Sensitivity DNA chip profile of sheared human gDNA:

smear around 200 bp.
The genomic DNA was diluted in GenDNA TE to reach a concentration of 100 ng/μl and 100
μl were sheared in a 0.65 ml Bioruptor Microtube (C30010011). The following program wasused: 13 Cycles [30 seconds “ON” & 30 seconds “OFF”], on Bioruptor® Pico sonication device(B01080010).

Spike-in recovery & MeDIP specificity
99.96%

100%

99.93%

75%

99.92%

71%

65%

50%
35%
25%

0%

0.05%

0.04%

0.01%
1µg

100ng
% recovery unDNA% recovery meDNA

10ng
Specificity

Figure 4. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after

IP. Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the
MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after
IP has been performed with IPure kit V2 (Diagenode).

48

MAGMEDIP KIT MANUAL

Figure 5. BioAnalyzer (Agilent) High Sensitivity DNA chip profile of IP (red) and corresponding
INPUT (blue) libraries.
Samples were prepared using 100 ng sheared human gDNA with the MagMeDIP kit(Diagenode) and a commercially available library preparation kit. DNA isolation after IP has
been performed with IPure kit V2 (Diagenode).

49

TSH2B

GADPH

Figure 6. Sequencing profiles of MeDIP-seq libraries prepared from different starting amountsof sheared gDNA on the positive and negative methylated control regions.
MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 µg (green),
50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit(Diagenode) and a commercially available library prep kit. DNA isolation after IP has been
performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples weresequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the humangenome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identicalscale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blueboxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone
variant that does not occur in blood cells, and it is known to be silenced by methylation.
Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows
the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The
GADPH gene is a highly active transcription region and should not be methylated, resulting in
no reads accumulation following MeDIP-seq experiment.

50

MAGMEDIP KIT MANUALFAQs
What is the resolution for MagMeDIP?
The resolution is related to the size of the fragments analyzed.
- For MagMeDIP-qPCR the sheared DNA is a smear of 50-1000 bp which means that theresolution is also 50-1000 bp.
- For MagMeDIP-seq the resolution is related to the size of the fragments incorporated
into the library and then effectively sequenced. While the sheared DNA is a smear
between 50-500 bp, after library preparation and IP we observed fragment size
between 50 bp and maximum 450 bp (thus libraries including adaptors around 200600 bp). On Illumina sequencers, clustering of libraries above 500 bp is not very
efficient, thus you can expect a resolution between 50 bp and 350 bp.
Would it be possible to obtain genomic DNA by other means and start using the kit at the
DNA shearing step? Can we clean up the DNA by column, to minimize the contact withphenol?
The XL GenDNA extraction module allows the extraction of high molecular weight DNA, but
you can definitely use columns instead of the double phenol/chloroform extraction, as longas you are confident with the quality of the genomic DNA you will get. The most important is
to obtain pure and high molecular weight DNA. You can choose which DNA extraction
protocol you want to use to reach this goal and then start using the MagMeDIP kit from theimmunoprecipitation step.
I will shear the DNA using the Bioruptor Pico. How do I choose the right tubes?
For shearing it is paramount to follow recommendations on appropriate volumes and tubes
according to your sample quantity. To help you with DNA shearing we have developed a tool
available at https://www.diagenode.com/en/dna-shearing-guide that contains all theinformation needed to choose the best shearing protocol for DNA.
Can I shear DNA using an enzyme for MagMeDIP DNA preparation?
As long as you obtain DNA fragments from 100-600 bp, you can use the fragmentation
technique of your choice. We usually do not recommend using restriction enzymes because
the fragmentation is less random compared to sonication.
Can I use more than 1 µg DNA for the MeDIP? Will it increase the overall yield?
It is possible to use more DNA, but the amount of antibody may need to be adjusted, requiring
further optimization without a guarantee of improved output. Therefore, we recommend
following the standard and optimized protocol. To increase the output we suggest to process
several samples with 1 μg and to pool them at the end.
51

Can I use another antibody with the kit?
This kit has been validated with the provided antibody only: 5-methylcytosine(5-mC)
Antibody - clone 33D3, Cat. No. C15200081, therefore we do not recommend changing theantibody. Moreover, not all antibodies are compatible with the beads which are included in
the kit.
What is the reference of the antibody used in the MagMeDIP kit? Which amount of antibody
per IP should I use?
The antibody used is the Cat. No. C15200081. To ensure efficient IP it is important to use thediluted antibody as described in the protocol. A lack of antibody can result in low IP efficiency
whereas a large excess of antibody might lead to lower specificity.
What are the internal controls that can be added to the incubation mix during MeDIP? Is thisa required step?
The internal spike-in controls allow you to check the immunoprecipitation efficiency of apositive and a negative template in the same tube as your DNA of interest. The purpose of
these controls is to confirm antibody specificity. It is better to add them to every sample, but
you can also choose to add a spike-in control to only one sample per experimental condition.
Is it possible to buy other spike-in controls tubes or primers? If possible, are they the same asin the initial MagMeDIP kit?
If you need more spike-in controls, you can buy the DNA Methylation Control Package V2(Cat. No. C02040019) which provides the methylated and unmethylated controls and theprimer pairs included in the MagMeDIP Kit. Those spike-in controls have been produced usingsynthetic oligonucleotides and are not homologous to any model species. Therefore, they will
not interfere with your DNA sample.
Another kind of methylated and unmethylated controls and primer pairs are available
separately in the DNA Methylation Control Package (Cat. No. C02040012). Those spike-in
controls are produced from the genome of A. thaliana and may therefore interfere with plant
samples.
Please check the products datasheet on the website for more information:
DNA Methylation Control
Package

52

Link towards datasheet

V1 (C02040012)

https://www.diagenode.com/files/products/kits/Datasheet_DNA_m
ethylation_control_package.pdf

V2 (C02040019)

https://www.diagenode.com/files/products/kits/Datasheet_DNAmethylation-control-package-V2.pdf

MAGMEDIP KIT MANUAL

What are the lengths of the PCR products?
Here are the lengths of the PCR products obtained with the different primer pairs:• unmethylated spike-in control: 110 pb• methylated spike-in control: 103 pb
At which step can I freeze my samples and stop safely?
Samples can be frozen at several steps of the protocol:• After DNA extraction (step 2)• After DNA shearing (step 3)• After isolation of IP’ed DNA (step 6)
How can I troubleshoot errors on the IP-Star Compact?
Regarding the questions related to the IP-Star Compact, please refer to the sections
Troubleshooting and list of error codes of the IP-Star Compact manual.

53

Related Products
Equipment

Cat. No.

DiaMag 0.2ml - magnetic rack

B04000001

Bioruptor® Pico sonication device

B01080010

Bioruptor® Plus sonication device

B01020001

Product

Cat. No.

5-methylcytosine (5-mC) monoclonal antibody (33D3)

C15200081

DiaMag anti-mouse IgG coated magnetic beads

C03010022

XL GenDNA Extraction Module

C03030020

DNA methylation control package V2

C02040019

DNA Methylation control package

C02040012

Human meDNA primer pair (TSH2B)

C17011041

Human meDNA primer pair (GAPDH)

C17011047

Mouse meDNA primer pair (TSH2B)

C17021042

Mouse meDNA primer pair (GAPDH)C17021045

Rat meDNA primer pair (TSH2B)

C17031043

Rat meDNA primer pair (GAPDH)

C17031046

IPure kit v2

C03010014

Revision history
VersionDate ofmodification

Version 5 05_2023

May 2023

Version 5 03_2023

March 2023

Version 5 12_2022

December 2022

Description of modifications
Page 8 - Adjustment of reagents quantity
Correction of small errors (point 4.15, 4.16, 4.17)
Removal of TSHB2 and GAPDH primer pairs from the list of kit components
and adjustment of the qPCR protocol
Adjustment of the auto protocol – the use of the IPure kit instead of DIB
Page 8 - Addition of the information about the modification of two buffers- Modification of the kit nameVersion 5 11_2022

54

MAGMEDIP KIT MANUAL

November 2022

- Removal of discontinued products- Adjustment of the recommendations and protocol for starting amount- Addition of the recommendations for primer design- Updating of the protocol for DNA shearing- Addition of the recommendations for MeDIP-seq- Addition of the table for antibody dilution

FOR RESEARCH USE ONLY.
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© 2023 Diagenode S.A. All rights reserved. No part of this publication may be reproduced, transmitted,
transcribed, stored in retrieval systems, or translated into any language or computer language, in any formor by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without priorwritten permission from Diagenode SA (hereinafter, “Diagenode”). The information in this guide is subject tochange without notice. Diagenode and/or its affiliates reserve the right to change products and services at
any time to incorporate the latest technological developments. Although this guide has been prepared withevery precaution to ensure accuracy, Diagenode and/or its affiliates assume no liability for any errors oromissions, nor for any damages resulting from the application or use of this information. Diagenodewelcomes customer input on corrections and suggestions for improvement.

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55

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