AUTO MeDIP KIT MANUAL

Cat. No. C02010011 (AF-Auto01-0016)

C02010012 (AF-Auto01-0100)

Version 7 I 02.14

Technical Assistance & Ordering Information

Diagenode s.a. BELGIUM | EUROPE

LIEGE SCIENCE PARK

Rue Bois Saint-Jean, 3

4102 Seraing - Belgium

Tel: +32 4 364 20 50

Fax: +32 4 364 20 51 [email protected]

[email protected]

Diagenode Inc. USA | NORTH AMERICA

400 Morris Avenue, Suite #101

Denville, NJ 07834 - USA

Tel: +1 862 209-4680

Fax: +1 862 209-4681 [email protected]

[email protected]

For a complete listing of Diagenode’s international distributors, visit: http://www.diagenode.com/en/company/distributors.php

For the rest of the world, please contact Diagenode s.a.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

SX-8G IP-Star Automated System for ChIP, MeDIP &MBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Kit Method Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Kit Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Kit Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

How to perform Automated MeDIP in the SX-8G IP-Star

®

Compact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Running a protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

How to perform Automated MeDIP in the SX-8G IP-Star

®

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Loading and running protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Shutting down the SX-8G IP-Star

®

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Quantitative PCR & Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Back Cover

PAGE 3 www.diagenode.com |

PAGE 4 DIAGENODE AUTO MeDIP KIT USER MANUAL

Introduction

The Diagenode SX-8G IP-Star

®

Automated System automates immunoprecipitation and increases reproducibility

Diagenode, the leading provider of complete solutions for epigenetics research, offers a variety of end-to-end systems to streamline DNA methylation and chromatin immunoprecipitation workflows. Central to this full offering is Diagenode’s

Automated Systems, simple yet robust automated bench-top instruments that standardize different epigenetic applications (i.e. ChIP, MeDIP or MethylCap). Diagenode designed these automation systems to make ChIP and DNA methylation studies accessible and reproducible, and ensure consistent data in every experiment.

Diagenode Automated Systems will produce consistent results from any operator regardless of the day, the experimental run, or the lab. Robust and reproducible results is a major goal of today’s high resolution epigenomic studies.

Diagenode Automated Platforms replace the numerous manual, error-prone steps of complex epigenetic applications with a reliable, highly consistent and automated process that requires minimal operator intervention. We empower researchers to simplify the tedious protocols and the complexity of many epigenetic protocols. In addition, Diagenode

Automated Systems minimize sample carryover, data variability, and costly errors. The platforms offer full workflow support for epigenetics research, utilizing our complete kits and laboratory-validated protocols to rapidly deliver highquality and consistent data.

Auto MeDIP kit

The Diagenode Auto MeDIP kit is designed to perform automated Immunoprecipitate of methylated DNA using the

SX-8G IP-Star.

The Auto MeDIP kit contains the antibody directed against 5-methyl Cytidine as well as meDNA and unDNA internal IP controls. The IP has been optimized to specifically select and precipitate the methylated DNA: by the use of our antibody, buffers and protocol. The IP efficiency can indeed be double-checked with the use of our internal controls. Furthermore, the use of the Automated System will drastically increase the consistency of your MeDIP Assay.

The Auto MeDIP kit allows you to perform DNA methylation analysis of your sample together with optimized internal IP controls: ALL IN ONE TUBE. The methylated DNA (meDNA) and unmethylated DNA (unDNA) controls allow for direct

CORRELATION between IP’d MATERIAL and METHYLATION STATUS. This methylation analysis is FAST, HIGHLY SPECIFIC and each IP is QUALITY controlled: essential keys for RELIABLE results.

In the Auto MeDIP kit, the protocol has been improved to allow researchers to work in smaller tubes than traditionally did so far. The kit ensures the use of low amount of reagents per reaction (not only antibodies, but also buffers) and it includes fewer buffers in comparison with other kits.

The Kit provides you with a DNA isolation buffer for an extra-fast method to purify your IP’d material (for qPCR analysis).

Alternatively, for other applications, e.g. sequencing, linear amplification or microarray. Magnetic DNA purification

(IPure) protocols can be used as alternative.

Combination of this High Quality Kit and the SX-8G IP-Star Robot will allow you to perform DNA Methyaltion Profiling in less than 9 hours! Starting with sheared DNA the Automated System will provide you with the purified methylated DNA of your sample.

The Auto MeDIP kit protocol has been validated using genomic DNA sheared by sonication using the Bioruptor®.

Innovating Epigenetic Solutions

SX-8G IP-Star

®

and SX-8G IP-Star

®

Compact Systems for automation of epigenetic applications

Diagenode has developed two automated platforms (SX-8G IP-Star

®

and SX-8G IP-Star

®

Compact) designed to increase your lab’s productivity, efficiency and experimental reproducibility. The two automated platforms are capable of processing up to 16 samples per cycle. The automated systems processes sheared chromatin (or DNA) to deliver purified DNA ready for qPCR, amplification, microarray and sequencing analysis. Both, the SX-8G IP-Star

®

and SX-8G IP-star

®

Compact have an easy-to-use open software that provides you with flexibility. This allows you to create your personal protocol according to your specific needs.

Major benefits of Diagenode Automated Platforms

SX-8G IP-Star

®

Compact SX-8G IP-Star

®

PAGE 5

> Increased reproducibility

> A consistency “Walk-away research” (less “hands-on-work”)

> Huge time savings

> Easy and flexible programming

> Validated for ChIP, MeDIP & MBD

> Compatible with Diagenode Kits (Auto ChIP kit, Auto Histone ChIP-seq kit, Auto Transcription ChIP kit, Auto

MeDIP kit, Auto MethylCap kit, Auto hMeDIP kit, Auto IPure kit)

> Reduces cross-contamination www.diagenode.com |

PAGE 6 DIAGENODE AUTO MeDIP KIT USER MANUAL

Applications

SX-8G IP-Star

®

Compact

ChIP-seq, MeDIP-seq, MethylCap-seq, hMeDIP, IPure, Sample preparation,

Re-ChIP, MagBisulfite, RNA-IP, Library preparation for NGS platforms.

SX-8G IP-Star

®

ChIP-seq, MeDIP-seq, MethylCap-seq, hMeDIP, IPure, Sample preparation, Re-ChIP,

MagBisulfite, RNA-IP.

Software

User interface

User friendly

Dispensing

Protocol optimization

(flexible parameters)

Intuitive touch screen panel

Software training not required

Automated dispension of assay reagents

PC Software

Software training before use

Manual dispension of assay reagents

Antibody coating (temperature, time, mixing speed)

Immunoprecipitation (temperature, time, mixing speed)

Washes (temperature, time, mixing speed)

Antibody coating (temperature, time)

Immunoprecipitation (temperature, time)

New protocol development

Achievable by Diagenode product specialist Achievable by customer after training

Characteristics

750W x 740 D x 610 H | 100 kg

8 Nozzles X-Y-Z axis | 4 – 95°C

1070W x 650 D x 780 H | 130 kg

8 Nozzles X-Y-Z axis | 4-95°C

Innovating Epigenetic Solutions

Improved reproducibility

100,0

80,0

60,0

100,0

40,0

80,0

20,0

60,0

40,0

20,0

Our SX-8G IP-Star will increase the immunoprecipitation reproducibility between IPs performed by the same as well as by different operators (see figure 1 and 2 below). Reagents (Antibodies, buffers,...) and sheared chromatin were identical for “ManChIP” and “AutoChIP”. The SX-8G IP-Star Automated system removes variation that can be created by manual handling and allows you to optimize and standardize your assay within a lab. The SX-8G IP-Star is designed to improve the accuracy and the reproducibility of any immunoprecipitiation experiment.

Man ChIP

SD(IgG)=0,69%

SD(H3K9me3)=23,84%

Man ChIP

SD(IgG)=0,94%

SD(H3K9me3)=11,36%

A

ChIP 1 ChIP 2

SD(IgG)=0,94%

SD(H3K9me3)=11,36%

A

34,63

ChIP 1 ChIP 2

1,96

34,63

0,63

50,70

SD(IgG)=0,17%

SD(H3K9me3)=1,12%

B

ChIP 1 ChIP 2

SD(IgG)=0,17%

SD(H3K9me3)=1,12%

B

56,25

ChIP 1 ChIP 2

1,86

57,83

1,62

56,25

SD(IgG)=1,4%

SD(H3K9me3)=2,38%

SD(IgG)=0,69%

SD(H3K9me3)=23,84%

C

ChIP 1 ChIP 2

SD(IgG)=1,4%

SD(H3K9me3)=2,38%

C

SD(IgG)=0,09%

SD(H3K9me3)=0,65%

D

ChIP 1

98,62

ChIP 2

95,26

SD(IgG)=0,09%

ChIP 2

SD(H3K9me3)=0,65%

D

44,75

2,06 1,42

ChIP 1

1,54

43,83

ChIP 2

1,42

44,75

Figure 1: Manual ChIP. Four different operators have each performed two ChIP experiments using H3K9me3 antibody on the genomic region SAT2 (positive locus). 10,000 Hela cells have been used per IP. Reagents and sheared chromatin were identical per assay. The standard deviations between the ChIPs performed by the same operator and between the four different operators are displayed.

1,96 0,63 1,86 1,62

2,06 1,42 1,54

1,42

Auto ChIP

Auto ChIP

90,0

80,0

ChIP 1

56,25

ChIP 1

56,25

30,0

20,0

10,0

1,26

IgG H3K9me3

1,26

IgG H3K9me3

ChIP 2

54,71

ChIP 2

54,71

1,00

IgG H3K9me3

1,00

IgG H3K9me3

ChIP 3

57,83

ChIP 3

57,83

1,45

IgG H3K9me3

1,45

IgG H3K9me3

SD(IgG)=0,28%

SD(H3K9me3)=1,6%

SD(IgG)=0,28%

SD(H3K9me3)=1,6%

ChIP 4

54,34

ChIP 4

54,34

0,81

IgG H3K9me3

0,81

IgG H3K9me3

Figure 2: Automated ChIP. Four ChIP experiments using H3K9me3 antibody on the genomic region SAT2 (positive locus) have been performed by the SX-

8G IP-Star. 10,000 Hela cells have been used per IP. Reagents and sheared chromatin were identical per assay. The standard deviations between the four

ChIPs performed by the SX-8G IP-Star are displayed.

PAGE 7 www.diagenode.com |

PAGE 8 DIAGENODE AUTO MeDIP KIT USER MANUAL

Kit Method Overview

Chromatin/DNA Shearing

(Bioruptor

®

Sonication)

Increased Reproducibility

Automated & High -Throughput

No “Foaming”

No Risk of Contamination

ST

EP

2

STEP 1

Chromatin/DNA Preparation

Chromatin Shearing

Optimization kit (Low SDS,

Medium SDS and High SDS)

Next Gen Sequencing

Bioruptor

®

Pico

Magnetic IP

Chromatin study

Auto True MicroChIP Kit

Auto Histone ChIP-Seq kit

Auto Transcription ChIP kit

DNA Methylation

Auto MeDIP Kit

Auto hMeDIP Kit

Auto MethylCap Kit

ST

EP

3

20

m in

Hands-on time

10 min

STEP 4

in

15 m

STE

P 5

DNA Purification

5 m in

ST

EP

6

Size Selection

with AMPure

®

XP beads

Library Preparation

Illumina

®

TruSeq™ ChIP

IPure kit

(magnetic purification)

DNA Isolation Buffer

NEBNext ® ChIP-seq

MicroPlex Library Preparation kit

(50 pg, multiplex, manual) qPCR

Figure 3. Diagenode provides a full suite of automated solutions for ChIP experiments.

For Step 1, we offer products to isolate nuclei and chromatin. Step 2 describes reproducible sample shearing with the Bioruptor and antibody capture needs.

®

product line. In Step 3 and Step 4, the Diagenode IP-Star Compact provides error-free, walk-away automation for all your immunoprecipitation

Innovating Epigenetic Solutions

Kit Materials

Kit Content

Two Auto MeDIP Kit formats are available and the kit content is sufficient to perform either 16 or 100 MeDIP assays by using the SX-8G IP-Star Automated System. The kit content is described in Table 1. Upon receipt, store the components at the temperatures indicated in Table.

Table 1. Kit content

Description

Magbeads

Water

MagBuffer A (5x)

MagBuffer B

MagBuffer C

Antibody anti-5meC meDNA Positive control unDNA Negative control

MagWash buffer-1

MagWash buffer-2

DNA Isolation Buffer (DIB)

Proteinase K

Primer pair #1 (meDNA control)

Primer pair #2 (unDNA control)

Quantity (x16)

250 µl

3 ml

3 ml

150 µl

60 µl

10 µl

40 µl

40 µl

10 ml

6 ml

6 ml

60 µl

50 µl

50 µl

Quantity (x100)

1.3 ml

20 ml

20 ml

1000 µl

400 µl

50 µl

40 µl

40 µl

60 ml

40 ml

40 ml

400 µl

50 µl

50 µl

Storage

4°C (do not freeze)

4°C

4°C

4°C

- 20°C

- 20°C (-80°C)

- 20°C

- 20°C

4°C

4°C

4°C

-20°C

-20°C

-20°C

Table 2. Components available separately

Description

200 µl tube strips (12 tubes/strip) + cap strips

200 µl tube strips (8 tubes/strip)

+ cap strips for SX-8G IP-Star

®

Compact

Tips (bulk)

Tips (box)

Reference

WA-001-0080

WA-002-0120

WC-001-1000

WC-002-0960

Quantity

80

120

1000

10x96

Storage

RT

RT

RT

RT

Table 3. Modules available separately

Description

XL GenDNA Extraction Module

Comments

For easy and fast

DNA extraction

Reference mc-magme-003

Quantity

60 rxns

PAGE 9 www.diagenode.com |

PAGE 10 DIAGENODE AUTO MeDIP KIT USER MANUAL

Table 3. Kits and Modules available separately

Description

Chromatin shearing optimization kit - Low SDS

Chromatin shearing optimization kit - Medium SDS

Chromatin shearing optimization kit - High SDS

IPure

Auto IPure

Table 4. Plastics and consumables available separately

Description

200 µl tube strips (12 tubes/strip) + cap strips

200 µl tube strips (8 tubes/strip) + cap strips for SX-8G IP-Star

®

Compact

96 well microplates

Tips (box)

Tips (bulk)

2 ml microtube for SX-8G IP-Star

®

Compact

Large reagent container for SX-8G IP-Star

®

Compact

Medium reagent container for SX-8G IP-Star

®

Compact

Reference

C01020010 (AA-001-0100)

C01020011 (AA-002-0100)

C01020012 (AA-003-0100)

AL-100-0100

AL-Auto01-0100

Reference

WA-001-0080

WA-002-0120

WA-003-0010

WC-002-0960

WC-001-1000

WA-008-0100

WA-007-0020

WA-006-0010

Quantity

1 kit

1 kit

1 kit

100 rxns

100 rxns

Quantity

80 pc

120 pc

10 pc

960 pc

1000 pc

100 pc

20 pc

10 pc

Innovating Epigenetic Solutions

How to perform Automated MeDIP in the SX-8G IP-Star

®

Compact

A. Prepare Reagents

1. Preparation of MagBuffer A (1x) (for 1 IP) in 1.5 ml tube.

MagBuffer (1x)

MagBuffer A (5x)

Water

200 µl

40 µl

160 µl

B. Prepare Antibody and IP Mixes

2. Preparation of Antibody Dilution (1:2) (max. 6 IP) in 1.5 ml tube.

Antibody (1:2)

Antibody

Water

2 µl

1 µl

1 µl

3. Preparation of Antibody Mix in 1.5 ml tube.

Antibody Mix

Antibody 1:2

MagBuffer A (5x)

Water

MagBuffer C

FINAL VOLUME

One IP

0.30 µl

0.60 µl

2.10 µl

2.00 µl

5.00 µl

For 2 IPs

0.75 µl

1.50 µl

5.25 µl

5.00 µl

12.50 µl

4. Preparation of Incubation Mix (1 IP + 1 Input) in 1.5 ml tube.

Incubation Mix

H

2

O

MagBuffer A (5x)

MagBuffer B meDNA positive control unDNA negative control

Sheared DNA (0.1 µg/µl)

90 µl

45 µl

24 µl

6 µl

1.5 µl

1.5 µl

12 µl

For 4 IPs

1.50 µl

3.00 µl

10.50 µl

10.00 µl

25.00 µl a. Incubate at 95°C for 3 minutes b. Quickly chill on ice (it is best to use ice-water) c. Perform a short spin at 4°C.

Note: The incubation mix is prepared in excess. 75 µl will be needed for the IP.

d. Take 7.5 µl incubation mix and store it in a new tube. This is the input.

Before dispensing, mix the content in the tube by pippeting up and down.

5. Preparation of Immunoprecipitation Mix (for 1 IP) in 1.5 ml tube.

Immunoprecipitation Mix

MagBuffer A (1x)

Incubation Mix

Antibody Mix

100 µl

20 µl

75 µl

5 µl

For 6 IPs

2.00

4.00

14.00

13.00

33.00

For 8 IPs

3.00 µl

6.00 µl

21.00 µl

20.00 µl

50.00 µl www.diagenode.com |

PAGE 11

PAGE 12 DIAGENODE AUTO MeDIP KIT USER MANUAL

Running a protocol

Diagenode Splash Screen – A0

After the software start-up screen disappears, the Diagenode splash screen is displayed for several seconds, and then disappears.

Start Screen – Top menu

After the Digenode splash screen disappears, the start screen is displayed. This is the first active window; it allows the user to enter into three different parts of the software.

USER ACTIONS:

Buttons:

• Protocols

• Maintenance (for technical services)

• Information (for Diagenode contact details)

Protocols screen

All available protocols are displayed on this screen.

Innovating Epigenetic Solutions

Keyboard

Screen – [Categories Name] Protocol List

After the user presses the “[Categories Name]” button, the “[Categories

Name]” appears. When selected the protocol on the protocol list, the

“Run” button shall turn executable.

Buttons:

• The user presses the “Back” button. The user returns to the

“Protocols” screen.

• The user presses the “Shutdown” button. The screen shall be changed to “Power Off”.

• The user presses the “Run” button. The screen shall be changed to “Sample number”.

• Page up the list box.

• Page down the list box

Screen – Sample number

After the user presses the “Run” button, the “Sample number” appears.

Buttons:

• The user presses the “Sample number” Text box. The screen will be changed to keyboard.

• The user presses the “Back” button. The user returns to the

“Protocol List” screen.

• The user presses the “Next” button. The screen shall be changed to “Configuration” or “Layout information”.

PAGE 13 www.diagenode.com |

PAGE 14 DIAGENODE AUTO MeDIP KIT USER MANUAL

Keyboard

Screen – Configuration

After the user presses the next button from the “Sample number” screen, the “Configuration” screen appears.

Buttons:

• The user presses the “Back” button. The user returns to the

“Protocol List” screen.

• The user presses the “Next” button. The screen shall be changed to “Layout information”.

• The user presses the “Save Parameter” button. The screen will be changed to “Save Parameter - Confirmation”.

- OK – Current parameters shown in the Display View will be stored to the [Protocol].ptd. And, returns the user to the display of the “Configuration” screen.

- No – Returns the user to the display of the “Configuration” screen.

• The user presses the Text box. The screen will be changed to

Keyboard or Speed list menu.

Speed list menu

Innovating Epigenetic Solutions

Layout information

Screen – Layout Information

After the user presses the “next” button from “Sample number” screen or

“Configuration” screen, the “Layout Information” screen appears.

Buttons:

• The user presses the “Back” button. The user returns to the previous screen.

• The user presses the “Next” button. The screen changes to “Set confirmation”.

• When the user presses a block, that block is magnifies on the work surface layout background. The magnified view provides a better display of the correct method setup for that block on the work surface.

• Based on the selected protocol, the user follows the indications provided in the screens to set up correctly the different reagents and samples based on the selected ChIP protocol.

Block-Tip

PAGE 15

Block-Regent Tip Rack

Block-PCR Tube www.diagenode.com |

PAGE 16 DIAGENODE AUTO MeDIP KIT USER MANUAL

Select a Protocol name

Input value in the

“Sample Number”

Input value in the

“Configuration”

Current Temperature Value

Screen – Set confirmation

After the user presses the “next” button in the “Layout information” screen, the “Set confirmation” screen appears.

At this point, user is expected to be ready to press RUN.

Buttons:

• The user presses the “Back” button. The user returns to the layout information screen.

• The user presses the “Run” button. This is the expected action when user gets to this display after reviewing blocks. Runs the protocol.

Protocol name

Progress Bar

Remaining time

Current Temperature Value

Screen – Running

After the user presses the “Run” button in the “Set confirmation” screen, the “Running” screen appears.

Buttons:

• The user presses the “Stop” button. The screen changes to “Stop Dialog”.

Status screen is preferred as a progress bar that moves across the screen as the step progresses

Innovating Epigenetic Solutions

A.

MeDIP - DIB

Screen – Running status

This screen gives informations about the current running step of the protocol.

The user can check through this screen the passed and remaining time of the experiment.

PAGE 17

B.

MeDIP - IPure

Screen – Elution

INPUT is defined as

INPUT= 7.5µl Incubation Mix + 92.5 µl

DIB buffer

IMPORTANT: Please note that the enriched methylated

DNA in DIB buffer is single strand DNA that can be directly analyzed by qPCR. For downstream applications such as sequencing or arrays, the enriched methyated

DNA needs to be purified by phenol/chroloform extraction and converted to double stranded DNA.

Screen – Elution

INPUT is defined as

INPUT= 7.5 µl Incubation Mix + 92.5 µl Elution

Buffer (IPure kit)

IMPORTANT: Please note that the enriched methylated DNA is single stranded DNA that needs to be purified before analysis.

DNA can be purified following IPure automated protocols and

Diagenode's Auto IPure kit (see Auto IPure kit user manual)

Alternatevely, the enriched methylated DNA could be purified by phenol/chroloform extraction or using spin columns. For downstream applications such as sequencing or arrays, the enriched methyated DNA needs to be converted to double stranded DNA.

Screen – Finish/End

When the protocol is complete, a window appears telling user the run is over. The screen behind this window should be the Startup screen.

When OK is pressed, then the Startup screen appears and the user can immediately begin to remove their sample and prepare the next run.

At this point, user is expected to be ready to press RUN.

Buttons:

• The user presses the “OK” button. Then screen shall be changed to “[Categories Name] Protocol List”.

www.diagenode.com |

PAGE 18 DIAGENODE AUTO MeDIP KIT USER MANUAL

A.

B.

Screen – Caution !

When the protocol finishes the user can return to the protocol list (screen A.) or warm the peltier block (screen B.) to eliminate possible condensation in the block.

Innovating Epigenetic Solutions

How to perform Automated MeDIP in the SX-8G IP-Star

®

A) Prepare Reagents

1. Preparation of MagBuffer A (1x) (for 1 IP) in 1.5 ml tube.

MagBuffer (1x)

MagBuffer A (5x)

Water

200 µl

40 µl

160 µl

B) Prepare Antibody and IP Mixes

2. Preparation of Antibody Dilution (1:2) (max. 6 IP) in 1.5 ml tube.

Antibody (1:2)

Antibody

Water

2 µl

1 µl

1 µl

3. Preparation of Antibody Mix in 1.5 ml tube.

Antibody Mix

Antibody 1:2

MagBuffer A (5x)

Water

MagBuffer C

FINAL VOLUME

One IP

0.30 µl

0.60 µl

2.10 µl

2.00 µl

5.00 µl

For 2 IPs

0.75 µl

1.50 µl

5.25 µl

5.00 µl

12.50 µl

For 4 IPs

1.50 µl

3.00 µl

10.50 µl

10.00 µl

25.00 µl

4. Preparation of Incubation Mix (1 IP + 1 Input) in 1.5 ml tube.

Incubation Mix

H

2

O

MagBuffer A (5x)

MagBuffer B meDNA positive control unDNA negative control

Sheared DNA (0.1 µg/µl)

90 µl

45 µl

24 µl

6 µl

1.5 µl

1.5 µl

12 µl

For 6 IPs

2.00

4.00

14.00

13.00

33.00

For 8 IPs

3.00 µl

6.00 µl

21.00 µl

20.00 µl

50.00 µl a. Incubate at 95°C for 3 minutes b. Quickly chill on ice (it is best to use ice-water) c. Perform a short spin at 4°C.

Note: The incubation mix is prepared in excess. 75 µl will be needed for the IP.

d. Take 7.5 µl incubation mix and store it in a new tube. This is the input.

Before dispensing, mix the content in the tube by pippeting up and down.

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PAGE 19

PAGE 20 DIAGENODE AUTO MeDIP KIT USER MANUAL

5. Preparation of Immunoprecipitation Mix (for 1 IP) in 1.5 ml tube.

Immunoprecipitation Mix

MagBuffer A (1x)

Incubation Mix

Antibody Mix

100 µl

20 µl

75 µl

5 µl

C) Dispense prepared reagents into the corresponding tubes (see picture below)

Note: Reagents dispension is different depending on the DNA purification method selected.

Loading reagents: make sure that all reagents are in the bottom of the tubes (especially magnetic beads) before starting the protocol.

Tube #

1

6

7

8

2

3

4

5

9

10

11

12

Description

DNA isolation buffer

Empty

Magnetic beads

MagBuffer A (1x)

MagBuffer A (1x)

-

Immunoprecitation Mix

MagWash buffer-1

MagWash buffer-1

MagWash buffer-1

MagWash buffer-2

DNA isolation buffer

DIB

Volume

92.5 µl

10 µl

50 µl

50 µl

100 µl

100 µl

100 µl

100 µl

100 µl

100 µl

Description

-

-

-

Elution buffer (IPure kit)

MagBuffer A (1x) + beads

MagBuffer A (1x)

Immunoprecitation Mix

MagWash buffer-1

MagWash buffer-1

MagWash buffer-1

MagWash buffer-2

Elution buffer (IPure kit)

IPURE

Volume

-

-

-

50 µl

50 µl + 10 µl

50 µl

100 µl

100 µl

100 µl

100 µl

100 µl

50 µl

1. For IPure method, Input sample will be purified following the IPure kit instructions.

2. For MeDIP-IPure experiments See intructions in the Auto IPure manual to prepare the Elution Buffer (Buffer A+ B)

DIB

DNA isolation buffer (Input)

Bead washes

Washes

1 2 3 4 5 6 7

MeDIP

8 9 10 11 12

DNA isolation buffer (IP)

IPure

1 2 3 4

Second elution and final sample position

Bead washes

MeDIP

Washes

5 6 7 8 9 10 11 12

First elution

IMPORTANT: At the end of the MeDIP-IPure reaction, 100 µl of IP sample are collected from well 4

Innovating Epigenetic Solutions

MeDIP protocols provided for the SX-8G IP-Star

DIB

8 IP's

16 IP's

Volumes

100 µl

100 µl

IPure

Loading and running protocol

Be sure that the computer connected to the robot never switches to the standby modus. (standby modus has to be inactivated). Standby of the computer will lead to the abort of the protocol.

1. Switch on the SX-8G IP Star. The power switch is on the right side of the instrument.

2. Switch on the computer.

3. Start SX-8G V52 software through SX-8G V52 the following icon

4. Place the prepared tube strip on the right cooling / heating block of the workstation

11

0

PAGE 21

5. Close the workstation door and lock it using the following icon

6. Press the following icon

Select the protocol of interest. Press start.

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PAGE 22 DIAGENODE AUTO MeDIP KIT USER MANUAL

IMPORTANT NOTE:

1. If the MeDIP protocols do not appear on the screen, Open the SX-8V52 directory and open Easy start ini file. Write the directory location of the protocols.

2. The Easy start ini file should contain the following information:

[EASYSTARTSCREEN]

HoldFilePath= C:\Documents and Settings\Desktop\New software protocols\ MeDIP

In red is indicated the the directory location of the MeDIP protocols

3. Start now SX-8G V52 software through SX-8G V52 exe.file

4. Press button for Easy Protocol Start screen and load the protocol of interest

Before starting the protocol a start confirmation window will appear. Press OK and the protocol will run.

Alternatively, temperature and incubation time for the IP reaction can be modified in an existing protocol by selecting the modify button. The modified protocol can be also saved as new protocol.

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7. The program will run through the following steps: magnetic bead washes, IP and IP washes.

During protocol the next window will be displayed indicating the current protocol step.

8. a) MeDIP-DIB

After the IP washes the following window will be appear.

PAGE 23

Follow the next instructions:

1. Add 7.5 µl of Incubation Mix (Input) to well 1

2. Add 1 µl proteinase K to wells 1 and 12

3. Close the tube strip with the corresponding caps

4. Press OK

7.5 µl input + 1 µl Proteinase K

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

IP DNA isolation

+ 1 µl Proteinase K

IMPORTANT: Please note that the enriched methylated in DIB buffer is single strand DNA that can be directly analyzed by qPCR.

For downstream applications such as sequencing or arrays, the enriched methyated DNA needs to be purified by phenol/chroloform extraction and converted to double stranded DNA.

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PAGE 24 DIAGENODE AUTO MeDIP KIT USER MANUAL

8. b) MeDIP-IPure

Follow the next instructions:

1. Collect samples from well 4 and keep them at 4 degrees

2. Follow instructions from the Auto IPure kit manual to proceed with the DNA purification of the samples and the input

IMPORTANT: Please note that the enriched methylated in DIB buffer is single strand DNA that can be directly analyzed by qPCR.

For downstream applications such as sequencing or arrays, the enriched methyated DNA needs to be purified by phenol/chroloform extraction and converted to double stranded DNA.

9. The following window will appear:

Close the workstation door and press OK.

The program will move forward to the next steps of the MeDIP protocol.

10. The SX-8G IP-Star software indicates the end of the protocol.

Collect your immunoprecipitated and isolated DNA

11. Discard magnetic beads by using the DiaMag02 (cat# kch-816-001) or by centrifugation.

12. This is your DNA ready for qPCR.

Shutting down the SX-8G IP-Star

1. Click on File and press End to close the software correctly.

2. Switch off the computer and its monitor.

3. Switch off the SX-8G IP-Star Automated System (power switch on the right side).

Note: Ensure that the door is closed!

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Quantitative PCR & Data Analysis

The Methylated DNA IP module includes four validated primer pairs specific to four types of DNA:

1) methylated DNA control (primer pair #1)

2) unmethylated DNA control (primer pair #2).

3) methylated human DNA region (testis-specific H2B, TSH2B)

4) unmethylated human DNA region (GAPDH promoter)

Note: Primer pairs for mouse and rat are available! Please visit www.diagenode.com

1. Prepare your qPCR mix using SYBR Green PCR master mix and start out qPCR. qPCR mix (total volume of 25 µl/reaction):

- 1.00 µl of provided primer pair (stock: 10 µM each: reverse and forward)

- 12.50 µl of master mix (e.g.: iQ SYBR Green supermix)

- 5.00 µl of isolated DNA or diluted purified DNA sample (see above for DNA dilutions)

- 6.50 µl of water

Table 1. qPCR cycles:

PCR

Amplification

Melting curve

Temperature

95°C

95°C

60°C

95°C

65°C and increment of 0.5°C per cycle

Time

7 minutes

15 seconds

60 seconds

1 minute

1 minute

Cycles x1 x40 x1 x60

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

• Data interpretation

The efficiency of methyl DNA immunoprecipitation of particular genomic locus can be calculated from qPCR data and reported as a recovery of starting material: % (meDNA-IP/ Total input).

% (meDNA-IP/ Total input)= 2^[(Ct(10%input) - 3.32) - Ct(meDNA-IP)]x 100%

Here 2 is the AE (amplification efficiency), Ct (meDNA-IP) and Ct (10%input) are threshold values obtained from exponential phase of qPCR for the methyl DNA sample and input sample respectively; the compensatory factor

(3.32) is used to take into account the dilution 1:10 of the input. The recovery is the % (meDNA-IP/ Total input).

• Background determination

The final goal of IP is to calculate the enrichment in the same IP sample of: 1/ the specific DNA fragments

(corresponding to the hydroxymethylated DNA) in comparison with 2/ non-methylated DNA (i.e. negative unDNA control).

• Relative occupancy can be calculated as a ratio of specific signal over background.

Occupancy= % input (specific loci) / % input (background loci)

Relative occupancy is then used as a measure of the hydroxymethylation of a specific locus; it provides clues about specificity of the IP. (background loci) corresponds to the signal obtained with one of the unmethylated DNA kit control.

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PAGE 25

PAGE 26 DIAGENODE AUTO MeDIP KIT USER MANUAL

Results

Automated Methyl DNA IP (9h)

Automated Methyl DNA IP (9h)

(IP reaction during 5h)

Automated Methyl DNA IP (19h)

(IP reaction during 15h)

Automated Methyl DNA IP (19h)

(IP reaction during 15h)

Figure 1: Automated MeDIP (9h)

IP reaction was performed with the anti-5meC antibody. Methylated and unmethylated DNA were used as internal controls. Unmethylated DNA region of GADPH and a methylated

DNA region of AlphaX1 were used to test DNA sample-IP efficiency.

DNA has been isolated by using DNA isolation buffer.

Figure 2: Automated MeDIP (19h)

IP reaction was performed with the anti-5meC antibody. Methylated and unmethylated DNA were used as internal controls. Unmethylated DNA region of GADPH and a methylated

DNA region of AlphaX1 were used to test DNA sample-IP efficiency.

DNA has been isolated by using DNA isolation buffer.

Troubleshooting Guide

Error Cause

SX-8G IP-Star cannot be switched on

Computer cannot be switched on

Remedy

SX-8G IP-Star is not receiving power. Check that the power cord is connected to the workstation and to the wall power outlet.

Computer is not receiving power. Check that the power cord is connected to the computer and to the wall power outlet.

SX-8G IP-Star is not switched on. Check that the SX-8G IP-Star is switched on.

SX-8G IP-Star shows no movement when a protocol is started

SX-8G IP-Star shows abnormal movement when a protocol is started

Aspirated liquid drips from the disposable tips

The pipettor head may have lost its home position. In the Software, select “Manual

Operation/Home”. After confirming that the pipettor head moves to the home position, run the protocol again.

Dripping is acceptable when ethanol is being handled. For other liquids: air is leaking from the syringe pumps. Grease or replace the O-rings. If the problem persists, contact DIAGENODE Technical Services.

Innovating Epigenetic Solutions

Technical Assistance

At DIAGENODE we pride ourselves on the quality and availability of our technical support. Our Technical Services

Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of DIAGENODE products. If you have any questions, or experience any difficulties regarding the SX-8G IP-

Star or DIAGENODE products in general, do not hesitate to contact us.

DIAGENODE customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at DIAGENODE. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques.

For technical assistance and more information call the DIAGENODE Technical Service Department or contact your local distributor.

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Ordering information

Description

SX-8G IP-Star

®

Compact

Auto True MicroChIP kit

Auto True MicroChIP & MicroPlex Library Prep Package

Cat. No. (NEW)

B03000002

C01010140

C01010141

Cat. No. (OLD)

UH-002-0001

/

/

MicroPlex Library Preparation kit x12

Auto Histone ChIP-seq kit protein A x16

Auto Histone ChIP-seq kit protein A x100

Auto Histone ChIP-seq kit prowwtein G x16

Auto Histone ChIP-seq kit protein G x100

Auto Transcription ChIP kit protein A x16

Auto Transcription ChIP kit protein A x100

Auto Transcription ChIP kit protein G x16

Auto Transcription ChIP kit protein G x100

Auto ChIP kit protein A x100

Auto ChIP kit protein G x100

Auto MeDIP kit x16

Auto MeDIP kit x100

Auto hMeDIP kit x16

Auto MethylCap x48

C05010010

C01010020

C01010022

C01010021

C01010023

C01010030

C01010032

C01010031

C01010033

C01010011

C01010013

C02010011

C02010012

C02010033

C02020011

AB-004-0012

AB-Auto02-A016

AB-Auto02-A100

AB-Auto02-G016

AB-Auto02-G100

AB-Auto03-A016

AB-Auto03-A100

AB-Auto03-G016

AB-Auto03-G100

AB-Auto01-A100

AB-Auto01-G100

AF-Auto01-0016

AF-Auto01-0100

AF-Auto02-0016

AF-Auto01-0048

Auto IPure kit C03010010 AL-Auto01-0100 100 rxns

Visit us at one of Diagenode’s demo sites or discover our Automated Systems by performing some assays with the help of our R&D and Technical Department.

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Format

1 unit

16 rxns

16 ChIP rxns & 12 library prep rxns

12 rxns

16 rxns

100 rxns

16 rxns

100 rxns

16 rxns

100 rxns

16 rxns

100 rxns

100 rxns

100 rxns

16 rxns

100 rxns

16 rxns

48 rxns

Diagenode s.a. BELGIUM | EUROPE

LIEGE SCIENCE PARK

Rue Bois Saint-Jean, 3

4102 Seraing - Belgium

Tel: +32 4 364 20 50 | Fax: +32 4 364 20 51 [email protected]

[email protected]

Diagenode Inc. USA | NORTH AMERICA

400 Morris Avenue, Suite #101

Denville, NJ 07834 - USA

Tel: +1 862 209-4680 | Fax: +1 862 209-4681 [email protected]

[email protected]

For a complete listing of Diagenode’s international distributors visit: www.diagenode.com/en/company/distributors.php

For rest of the world, please contact Diagenode s.a.

© 2013 Diagenode, Inc. All rights reserved. The content of this document cannot be reproduced without prior permission of the authors. Bioruptor and IP-Star are registered trademarks of Diagenode.

MA-AMeDIP-V7_02_14