hMeDIP kit
Hydroxymethylated DNA
Immunoprecipitation Kit
Cat. No. C02010031

USER GUIDE
Version 4

09_2022

Please read this manual carefully
before starting your experimentContents
Introduction ............................................................................................................... 4
Kit method overview .................................................................................................. 5
Kit Materials ............................................................................................................... 6
Required Materials Not Provided ............................................................................... 7
Remarks before starting ............................................................................................. 9
Protocol ..................................................................................................................... 9
STEP 1: Binding antibodies to hydroxymethylated DNA and bead preparation ............................. 13
STEP 2: Magnetic immunoprecipitation ......................................................................................... 15
STEP 3: DNA isolation ..................................................................................................................... 16
STEP 4: Quantitative PCR & Data analysis ...................................................................................... 17

Results ..................................................................................................................... 19
Related products ...................................................................................................... 20
Revision history ........................................................................................................ 22

Introduction
One of the fastest growing fields in biology and cancer research is epigenetics. While the underlying
genetic code defines which proteins and gene products are synthesized, it is epigenetic control thatdefines when and where they are expressed. Epigenetic control is generally mediated by methylationof cytosine to 5-methylcytosine in CpG islands and post-translational modification of histones.
Methylation of CpGs near promoters is classically associated with gene silencing or down-regulation,
as is deacetylation of histones.
Cytosine hydroxymethylation was recently discovered as another important epigenetic mechanism.
This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC)
into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5hmC is the subject of intense research and debate, early studies strongly indicate that it is also
involved in gene regulation and in numerous important biological processes including embryonicdevelopment, cellular differentiation, stem cell reprogramming and carcinogenesis.
The Diagenode hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented
genomic DNA samples for use in genome-wide methylation analysis. It features a highly specificmonoclonal antibody against 5-hmC for the immunoprecipitation (IP) of hydroxymethylated DNA.
Recovered DNA is suitable for many downstream applications to analyze genome-wide DNA
hydroxymethylation including PCR, sequencing, and microarrays. The IP efficiency can be monitoredwith the use of control DNA sequences (5-hmC and unmodified cytosine DNA standards) and control
primer pairs (internal and external controls). Performing hydroxymethylation profiling with hMeDIP
kit is FAST, RELIABLE and HIGHLY SPECIFIC.
The kit includes:•

All the reagents for the immunoprecipitation, including monoclonal antibody against 5-hmCand magnetic beads•

5-hmC and unmethylated spike-in controls and their associated qPCR primer pairs to verify
the efficiency of the IP•

qPCR primer pair against Sfi1 targeting hydroxymethylated gene in mouse to monitor the
success of the IP on your DNA samples•

DNA isolation module (after IP): 45 minutes for DNA Isolation Buffer method (DIB)

The kit ensures the use of a low quantity of antibodies and buffers per reaction. The number of steps
is reduced and handling is easier which makes this kit cost effective and simple to work with.
We recommend Diagenode’s Magnetic Rack (Cat. No. B04000001) together with our hMeDIP 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 the reaction volumes and use of reagents. The protocolhas been validated with Diagenode's Bioruptor® (Cat. No. B01060010 or B01020001). Nevertheless,
DNA can be sheared with any in house protocol and sonication devices as long as efficiency is checked
before use. This kit is provided with a manual protocol as well as an automated version for Diagenode
IP-Star® Compact Robot (Cat. No. B03000002).
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MAGMEDIP-SEQ PACKAGE V2 MANUAL

1. Cell collection and lysis

2. DNA Extraction and Shearing using Bioruptor®

PROTOCOL

Kit method overview

3. Immunoprecipitation, washes and DNA isolation

a.
b.
c.
d.

Add magnetic beads coated with antibody of interest
Magnetic capture of Antibody – DNA complex
Washes
DNA isolation before qPCR

5

Kit Materials
The kit content is sufficient to perform 16 hydroxymethylated DNA immunoprecipitations (hMeDIPs).
1. The kit content is described in Table 1. Upon receipt, store the components at thetemperatures indicated in Table 1.
Table 1. Components of the hMeDIP kit
Component

Quantity

StoragehMeDIP buffer H1

Ion chelator mix included (10x).

3 ml

4°C

hmeDNA control

0.001 ng/µl

40 µl

-20°C

unDNA control

0.1 ng/µl

15 µl

-20°C

5-hmC monoclonal antibody(mouse)Mouse IgG

1 µg/µl

32 µl (50 µg)-20°C

1 µg/µl

50 µl-20°C/4°C

220 µl4°C
Do not freeze

12 ml

4°C

hMeDIP buffer H2

The beads are supplied for 16 IPs;
detergent and 0.02%, sodium azide
included.
BSA and Ion chelator mix included.

hMeDIP buffer H3

Ion chelator mix included.

4 ml

4°C

DNA isolation Buffer (DIB)

-

5 ml

4°C

Proteinase K

100 x stock solution.

40 µl- 20°ChmeDNA primer pairs

5 μM each (Rv & Fw)

50 µl- 20°C

unDNA primer pairs

5 μM each (Rv & Fw)

50 µl- 20°C

Mouse Sfi1 primer pairs

5 μM each (Rv & Fw)

50 µl- 20°CPCR tube strips

For 1 row of 8 samples each.

4

RT

PCR tube caps

For 1 row of 8 samples each.

4

RT

Water

-

24 ml

4°C

Anti-mouse IgG coatedmagnetic beads

6

Comments

MAGMEDIP-SEQ PACKAGE V2 MANUALReagents for the IP and qPCR analysis•

Gloves to wear at all steps•

Autoclaved tips•

RNase/DNase-free 1.5-ml (or 2-ml) tubes•

PCR tubes and reagents•

Ultra pure water

PROTOCOL

Required Materials Not Provided

Equipment for the IP and qPCR analysis•

DiaMag 0.2ml - magnetic rack (Diagenode, Cat. No. B04000001)•

Centrifuges for 1.5-ml tubes (4°C)•

Rotating wheel (4°C)•

Vortex•Thermomixer (55°C, 95°C)•

Quantitative PCR facilities

Reagents and equipment for the DNA preparation and shearing•

Tubes: 1.5-ml and 50-ml conical tubes•

Trypsin-EDTA•

Ice-cold PBS buffer•

Agarose and TAE buffer•

DNA molecular weight marker•

Centrifuges for 1.5-ml tubes and 50-ml conical tubes (4°C)•

Agarose gel apparatus•

Cell counter•

Bioruptor Pico (Diagenode, Cat. No. B01060010) or the Bioruptor Plus (Diagenode, Cat. No.
B01020001)

7

Additionnal supplies (included and available separately)
Component

Cat. No.

Format5-hmC monoclonal antibody (mouse)

C15200200

DiaMag anti-mouse IgG coated magnetic beads

C03010022

Mouse IgG antibody

C15400001

100 µgMouse Sfi1 primer pairs

C17021046

50 µl

Proteinase K

C06050001
C06050002

100 µl250 µl20 µg/µl
50 µg/µl
100 µg/µl
220 µl660 µl
1.5 mlOptional supplies (not included and available separately)

8

Component

Cat. No.

FormatXL GenDNA Module

C03030020

6 rxns

Mouse gapdh promoter primer pair

C17021045

50 µl
500 µl5-hmC monoclonal antibody (rat)C15220001

20 µg
50 µg
100 µg5-hmC polyclonal antibody (rabbit)

C15410205

20 µg
50 µg

DiaMag protein G-coated magnetic beads

C03010021

220 µl660 µl
1.5 ml

DiaMag protein A-coated magnetic beads

C03010020

220 µl660 µl
1.5 mlRat IgG

C15420001

25 μl/µg

Rabbit IgG

C15410206

250 μl/µg

DNA hydroxymethylation control package

C02040018

48 rxns

DNA methylation control package V2

C02040019

48 rxns

IPure kit v2

C03010015

100 rxns

DiaMag 0.2ml - magnetic rack

B04000001

MAGMEDIP-SEQ PACKAGE V2 MANUALDNA extraction

The quality of the DNA to be used in hMeDIP is important. Thus, we recommend using Diagenode XLGenDNA Extraction Module (Cat. No. C03030020) 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.

PROTOCOL

Remarks before starting

The XL GenDNA Extraction Module for gDNA isolation provides sufficient volume of reagents for the
preparation of 6 gDNA batches, each obtained from 1 to 1.5 million cultured cells.

Starting amountConcentrations of all the reagents in the hMeDIP Kit are optimized for a starting amount of 1 μg ofsheared DNA per IP.

DNA shearing
For an efficient and best resolution IP experiment, the gDNA has to be sheared into fragments around
400 bp on the Bioruptor Pico (Cat. No. B01060010) or on the Bioruptor Plus (Cat. No. B01020001).
Before starting with IP experiment, we recommend analyzing the size of the fragments after shearing
using a dedicated device such as the BioAnalyzer (Agilent) or the Fragment Analyzer (Agilent).
Example of shearing for hMeDIP using the Bioruptor® Pico
The genomic DNA was diluted in TE buffer to reach a concentration of 100 ng/μl and 100 μl weresheared in a 0.65 ml Bioruptor® Microtube (Cat. No. C30010011). The following program was used:• Cycles: [15 seconds "ON" & 90 seconds "OFF"]• 8 cycles

Figure 1: Agilent High Sensitivity DNA chip profile of sheared genomic DNA: smear around 400 bp.

9Antibody choice

The hMeDIP kit includes a 5-hmC monoclonal mouse antibody (Cat. No. C15200200). However, it has
been also validated with a 5-hmC monoclonal rat antibody (Cat. No. C15220001) and a 5-hmC
polyclonal rabbit antibody (Cat. No. C15410205). Those antibodies can be used in combination withthe hMeDIP kit, as long as the magnetic beads and the negative controls are replaced accordingly.
Please note that:• the rat antibody (Cat. No. C15220001) should be used in combination with DiaMag protein
G-coated magnetic beads (Cat. No. C03010021) and rat IgG (Cat. No. C15420001) as negative
control.

• the rabbit antibody (Cat. No. C15410205) should be used in combination with DiaMag protein
A-coated magnetic beads (Cat. No. C03010020) and rabbit IgG (Cat. No. C15410206) as
negative control.

Magnetic beads

This kit includes magnetic beads (Magbeads). Please make sure the beads do not dry out during theprocedure as this may result in reduced performance. Keep the Magbeads homogenous insuspension at all times when pipetting. Variation in the number of beads will decrease reproducibility.
Do not freeze the Magbeads.

Hydroxymethylated and unmethylated spike-in controls

The hMeDIP Kit contains one hydroxymethylated and one unmethylated spike-in controls that can be
added directly to DNA samples before the IP. Those spike-in controls are not homologous to any
model species and will not interfere with the sample of interest. To check the efficiency of thehMeDIP experiment, this kit also includes primer pairs targeting a positive region on mouse genome
to calculate their recovery by qPCR. These primer pairs are only suitable for mouse
samples.

Technique for DNA isolation

The hMeDIP Kit includes a DNA Isolation Buffer for an easy and very fast DNA isolation after hMeDIP
providing DNA suitable for qPCR analysis. In case DNA of higher purity is needed for Next-Generation
Sequencing (NGS) or other downstream application than PCR, we recommend using the IPure kit v2(Cat. No. C03010014) for purification.

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MAGMEDIP-SEQ PACKAGE V2 MANUAL

Designing your primers

Self-complementarity and secondary structure of the primers can be tested for primer design(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Annealing temperature of 60°C is
recommended for qPCR primers.
Short length of amplified DNA fragment (50 - 100 bp) facilitates the PCR efficiency and reduces
potential problems in amplification of G/C-rich regions.
Difference in melting temperature between forward and reverse primers should not exceed 2 to
3°C.
G/C stretches at the 3’ end of the primers should be avoided.

PROTOCOL

PCR experiment preparation

Advantages of the qPCR
qPCR or Real time PCR enable fast, quantitative and reliable results.
The Gene Quantification page (www.gene-quantification.de) describes and summarises all technicalaspects involved in quantitative gene expression analysis using real-time qPCR & qRT-PCR. It
presents a lot of applications, chemistries, methods, algorithms, cyclers, kits, dyes, analysis
methods, meetings, workshops, and services involved.

Validation of your primers

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 by formula (1): AE= 10^(-1 / slope)
The ideal amplification factor is 2. If it is not the case the qPCR reagents from different brand or new
primers should be tested.

11

12

STEP 1 – Binding antibodies to hydroxymethylated DNA & bead preparation

13

STEP 2 – Magnetic immunoprecipitation

15

STEP 3 – DNA isolation

16

STEP 4 – Quantitative PCR & Data analysis

17

MAGMEDIP-SEQ PACKAGE V2 MANUALSTEP 1

Binding antibodies to hydroxymethylated DNA
and bead preparation

PROTOCOL1.

DAY 1: Binding antibodies to hydroxymethylated DNA
1.1

Prepare the IP incubation mix w/o the antibody and w/o magnetic beads for all yourhMeDIP reactions (Table 2).
NOTE: Make sure when working with hMeDIP buffer H1, that there are no crystals left insolution. Otherwise heat up
Table 2: IP incubation mix with no antibodies and no beads
Reagent

Volume per IP + INPUT

Volume per additional IP

Water

93.9 μl78.25 μlhMeDIP buffer H1

12 μl

10 μl

hmeDNA control

1.5 μl1.25 μlunDNA control

0.6 μl

0.5 μlDNA sample (0.1 μg/μl)*

12 μl

10 μl

TOTAL VOLUME

120 μl100 μl* If the DNA sample is at a concentration of 0.1 μ g/μl, use 93.9 μl water per IP. If the concentration of the DNA
sample is not at 0.1 μg/ μl, adjust the volumes of DNA and of water to add. Keep the volume of the incubation
mix at 120 μ l per IP+input. Caution: in any case 1 μg of DNA is needed per IP!

1.2

Incubate at 95°C for 10 minutes.

1.3

Quickly chill sample on ice (it is best to use ice-water).

1.4

Perform a pulse spin to consolidate your sample.

1.5

First, take out 10 μl per INPUT (that is 10% input) and transfer to a new labeled tube.
•

Keep the input samples at 4°C. The input sample is to be used as a control of starting
material and it is therefore not to be used in IP.

1.6

Transfer from what is left: 100 μl per tube into 200-μl tubes using the provided 200-μl
tube strips (or individual 200-μl tubes that can fit in our Magnetic Rack).

1.7

Add 2.5 μl of the 5-hmC antibody or 2.5 μl of Mouse IgG per tube.
•

1.8

The IgG is a negative control antibody. We recommend to include one IgG control foreach serie of hMeDIP reactions.

Incubate on a rotating wheel (40 rpm) at 4°C for 2 hours. During this time proceed to thebeads preparation.
13

Beads preparation
1.9

1.10

Wash the magnetic beads with ice-cold hMeDIP buffer H2 as follows: In 1.5 ml tube, add
200 μl of hMeDIP buffer H2 to 12 μl of beads and resuspend the beads. Incubate on arotating wheel (40 rpm) at 4ºC for 2 hours.
•

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

•

10 μl of beads are needed per IP.

After washing, briefly spin the tube containing the beads to bring down liquid caught in
the lid. Pellet the beads, discard the supernatant and keep the bead pellet (see below two
options to pellet the beads).
Options:a) Use the magnetic rack for 1.5 ml tubes.
b) Centrifuge for 5 minutes at 1,300 rpm.

1.11

In a new tube, dilute 1:10 in water, the provided hMeDIP buffer H1 to have the diluted
hMeDIP buffer H1:10.
•

1.12

Resuspend the beads in diluted hMeDIP buffer H1:10 to the volume then originally used.
•

14

350 μl of hMeDIP buffer H1:10 are needed per IP.

for 1 IP: 12 μl of hMeDIP buffer H1:10

MAGMEDIP-SEQ PACKAGE V2 MANUAL

Magnetic immunoprecipitation
2.1

Briefly spin the 8-tube strip containing the incubation mix with antibody or control (fromPoint 1.8) and add 10 μl of washed beads per tubes (from Point 1.12).

2.2

Incubate on a rotating wheel (40 rpm) at 4ºC for overnight.

PROTOCOL

STEP 2DAY 2
2.3

Place the 8 tubes strip in the DiaMag 0.2ml - magnetic rack - (Cat. No. B04000001), wait
1 minute and discard the buffer.

2.4

Wash three times using 100 μl of ice-cold hMeDIP buffer H1:10. Each wash is done asfollows: add buffer, close the tube caps, invert the 8-tube strip to resuspend the beads,
incubate for 5 minutes at 4°C on a rotating wheel (40 rpm), spin, place in the magnetic
rack, wait 1 minute and discard the buffer. Keep the captured beads.

2.5•

Do not disturb the captured beads attached to the tube wall.

•

Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning in
the magnetic rack.

Wash one time with 100 μl ice-cold hMeDIP buffer H3 (as described above: Point 2.4), spin,
place in the Magnetic Rack, wait 1 minute and discard the buffer. Keep the captured beads
for Point 3.3.

15

STEP 3

3.

DNA isolation

NOTE: This kit includes a DNA isolation buffer (DIB) for easy and very fast DNA isolation after hMeDIP

providing DNA suitable for qPCR analysis (STEP 4).
If you need DNA of higher purity for next generation sequencing or other downstream application than
PCR, we recommend using the IPure kit v2 (Cat. No. C03010014) for purification.

16

DNA isolation buffer (DIB)

IPure v2Time

45 minutes

1h30

DNA concentration+++ (possible to concentrate)

DNA purity+++

Subsequent analysis

qPCR

Next generation sequencing,
microarray, qPCR amplification

3.1

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

3.2

Prepare 100 μl complete DIB per sample as follows. Add 1 μl of Proteinase K per 100 μ l of
DIB. Scale accordingly knowing that 100 μl are needed per IP’d DNA sample and 90 μl, per
input DNA sample.

3.3

Remove the tubes from the magnetic rack and add 100 μ l of complete DIB buffer per IP’dDNA sample. Resuspend the beads.

3.4

Add 90 μl of complete DIB to 10 μl of input DNA sample.

3.5

Incubate at 55°C for 15 minutes both IP’d DNA sample and input DNA sample.

3.6

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

3.7

Label new 1.5 ml tubes:

3.8

Place the samples in the magnetic rack and wait 1 minute or until the supernatant is clear.

3.9

Transfer the supernatants in new labeled tubes. That is the DNA ready for qPCR analysis.
Store at -20°C.

MAGMEDIP-SEQ PACKAGE V2 MANUALPROTOCOLSTEP 4

Quantitative PCR & Data analysis
This last step consists in amplifying and analysing the IP’d DNA.
4.1

Prepare your qPCR mix using SYBR® PCR Green master mix and start out qPCR.
dqPCR mix (total volume of 25 μl/reaction):- 6.50 μl of water- 12.50 μl of master mix (e.g.: iQ SYBR® Green supermix)- 1.00 μl of provided primer pair (stock: 5 μM each: reverse and forward)- 5.00 μl of isolated DNA or INPUTPCR Amplification

Melting Curve

4.2

Temperature

Time

Cycle

95°C
95°C
60°C
95°C
65°C and increment of 0.5°C
per cycle

7 minutes
15 seconds
60 seconds
1 minutex1
x40

1 minutex1
X60

When the PCR is done, analyse the results. Some major advices are given below.

Data interpretation
The efficiency of hydroxymethyl DNA immunoprecipitation of particular genomic locus can becalculated from qPCR data and reported as a percentage of starting material: % recovery(hmeDNA-IP/ Total input).
% recovery = 2^[(Ct(10%input) - 3.32) - Ct(hmeDNA-IP)]x 100%

Here 2 is the AE (amplification efficiency); Ct (hmeDNA-IP) and Ct (10%input) are thresholdvalues obtained from exponential phase of qPCR for the hydroxymethyl DNA sample and input
sample respectively; the compensatory factor (3.32) is used to take into account the dilution
1:10 of the input.

17

Background determination
The final goal of IP is to calculate the enrichment in the same IP sample of the specific DNA
fragments (corresponding to the hydroxymethylated DNA) in comparison with background(corresponding to unmethylated DNA).
This enrichment can be calculated as a ratio of specific signal over background.
enrichment = % recovery (specific locus) / % recovery (background locus)

18

MAGMEDIP-SEQ PACKAGE V2 MANUAL

Results
hMeDIP results obtained using the hMeDIP kit (mouse monoclonal antibody), including:• 5-hmC mouse monoclonal antibody• 5-hmC and cytosine spike-in controls• Mouse IgG

hMeDIP-qPCR on the spike-in controls
90
80
70

% Recovery

60
50
40
30
20
10
0

IP 1

IP 2

IgG 1

IP 1

Blood DNA

IP 2

IgG 1

Colon DNA
hmeDNA

unmehylated spike-in

hMeDIP-qPCR was performed using the hMeDIP kit (mouse monoclonal antibody)
One μg of gDNA from human whole blood and colon has been sonicated with Bioruptor to obtainDNA fragments of 300-500 bp. The hMeDIP has been performed using Diagenode hMeDIP kit andprotocol in duplicates. The IgG isotype antibody from mouse was also included as negative control.
Unmethylated and hydroxymethylated spike-in controls have been added to the samples and thenanalyzed by qPCR using specific corresponding primer pairs.

19Related products

20

Product

Cat. No.

5-hmC monoclonal antibody (rat)C15220001

5-hmC polyclonal antibody (rabbit)

C15410205

DiaMag protein G-coated magnetic beads

C03010021

DiaMag protein A-coated magnetic beads

C03010020

Rat IgG

C15420001

Rabbit IgG

C15410206

MagMeDIP kit

C01020021

XL GenDNA Extraction Module

C03030020

DNA hydroxymethylation control package

C02040018

DNA methylation control package V2

C02040019

DiaMag 0.2ml - magnetic rack

B04000001

DiaMag rotator

B05000001

Bioruptor® Pico sonication device

B01060010

Bioruptor® Plus sonication device

B01020001

MAGMEDIP-SEQ PACKAGE V2 MANUAL

21

Revision history
VersionDate of modification

Description of modifications-

22

Version 4 09_2022

September 2022

Version 4 03_2022

March 2022

MAGMEDIP-SEQ PACKAGE V2 MANUAL-Correction of the antibody
concentration in the table of content
Removal of discontinued productRemoval of mDNA control andmodification of the corresponding
protocol (Step 1.1)

Page 7 - Correction of the storage temp. hMeDNA control

FOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
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Technologies Corporation.

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