Download - Diagenode

Reliable ChIP-seq results with the Diagenode True MicroChIP Kit and
MicroPlex Library Preparation Kit on only 10,000 cells
Céline Sabatel, Irina Panteleeva, Miklos Laczik, Geoffrey Berguet, Ignacio Mazon*, Sharon Squazzo*, Hélène Pendeville and Dominique Poncelet
Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium I *Diagenode inc., 400 Morris Avenue, Suite 101, Denville, NJ 07834, USA
MicroPlex library preparation kit protocol overview
Automated ChIP
The Diagenode True MicroChIP Kit has been developed with both an optimized protocol and reagents to
enable successful ChIP on 10,000 cells. Moreover, the True MicroChIP kit protocol has been thoroughly
optimized by Diagenode for ChIP followed by high-throughput sequencing on an Illumina® sequencer.
In addition, to enable sequencing on the low amounts of DNA recovered after ChIP on 10,000 cells,
Diagenode has developed a library preparation protocol for limited quantities of DNA. The MicroPlex
Library Preparation Kit requires only picogram amounts of ChIP’d DNA inputs for library preparation
compatible with the Illumina® platforms.
1. 6X faster than TruSeq
„„
„„
„„
„„
Add Pre-Mix
Add Pre-Mix
3 incubations in a single tube
No intermediate purifications
No transfers and no columns
2 Hours
Manual throughput up to 192 samples per day with one operator
dsDNA template
Cleavable
Replication
Stop
x
x
In this poster, we demontrated the successful use of the Diagenode True MicroChIP Kit in combination
with the MicroPlex Library Preparation Kit for ChIP-seq on the Illumina® platform.
+
Blunt-end Ligation
Nick
Nick
Introduction
Stem-loop
Adaptor
Extension and Cleavage
EIF4A2p
GAPDHp
MB2
Sat2
10
4. Greater coverage of complex
sequences
0
5. Automatable, indexed
H3K4me3
IgG
Manual ChIP
40
50 pg to 50 ng of fragmented ds DNA are converted into
sequencing-ready libraries for Illumina® NGS platforms
using a fast, simple and sensitive 3-step protocol. After
amplification, libraries are purified with AMPure XP
beads. The purified libraries are ready for sequencing
on an Illumina® platform. Indexing reagents included in
the kit allow the multiplexing of 12 samples in a single
sequencing lane.
Cleavable
Replication
stop
Addition of Adaptors
+
3. Simple preps
Figure 2. MicroPlex library preparation workflow.
DNA Repair
Stem-loop
Adaptor
2. 100X more sensitive
%input
Mix Reagent
and sample
NGS
Template
%input
Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the
gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP
protocols require abundant amounts of starting material (at least hundreds of thousands of cells per
immunoprecipitation) limiting the application for the ChIP technology to few cell samples.
20
TM
EIF4A2p
GAPDHp
MB2
Sat2
20
0
H3K4me3
4. Library preparation on low amount of ChIP’d DNA with no pre-amplification step using the MicroPlex kit
Figure 6. Library generation with the
MicroPlex kit.
A.
ChIP assays were performed on
10,000 and 100,000 HeLa cells with
Diagenode H3K4me3 antibody (0.25 µg/
reaction). Libraries were made with
the MicroPlex Library Preparation
Kit. The generated libraries were then
analysed on an Illumina® HiSeq2000.
Cluster generation and sequencing
were performed according to the
manufacturer’s instructions.
Barcode
The True MicroChIP Kit has been developed specifically for ChIP with limited cell numbers by optimizing
several parameters; such as:
Primer B
Results
•Chromatin shearing
•Antibody concentration
•Use of different carriers
•Stringency of washes
1. Efficient and easy chromatin shearing using Bioruptor® Pico and True MicroChIP kit Shearing Buffer
Figure 3. HeLa cells were fixed with 1% formaldehyde (for 10 minutes at RT). Cell lysis was performed using
the Lysis Buffer of the Diagenode True MicroChIP kit. Samples corresponding to 10 000 cells were sheared
during 5 rounds of 5 cycles of 30 seconds “ON” / 30 seconds “OFF” with the Bioruptor ® Pico. Samples were
vortexed before and after performing 5 sonication cycles, followed by a short centrifugation at 4°C. 10 μl of
DNA (equivalent to 60,000 cells) were analysed on a 1.5% agarose gel.
The MicroPlex Library Preparation Kit is specifically designed for library preparation. It requires only
3 steps with a single purification. The kit requires only picogram amounts of DNA as starting material,
allowing the sequencing of the low DNA amounts recovered after ChIP on low cell numbers.
B.
Methods
98%
ChIP-seq workflow
Repair
Repair
Clean-up
Ligate
antibodies
ChIPH3K4me3
H3K4me3
ChIP
14
14
A-Tail
Ligate
Clean-up
Gel purify
Clean-up
NGS-READY DNA
< 3 hours
1 tube
(3)
b.
a.
(4)
b.
c.
(5)
c.
d.
(6)
50 pg - 50 ng DNA input recommended
1 user-supplied reagent
22 pipetting steps
No sample transfers and no columns
1 clean-up at end
ChIP
ChIPH3K4me3
H3K4me3
192 sample throughput per day per tech
1414
d.
1212
b.
Figure 1
c.
8 8
00
8 8
True MicroChIP procedure: 1. Cell fixation and DNA-protein cross-linking 2. Cell lysis and
6 6 chromatin shearing using the
®
4 4
Bioruptor Pico 3. Binding of the antibodies to the chromatin.
Addition of beads. 4. Magnetic4immunoprecipitation
5. Washes
4
of immune complexes 6. DNA purification and recovery of ChIP’d DNA
ChIP-sequencing procedure: Comparison of the library preparation procedure using the MicroPlex kit and the TruSeq kit
0 0
0 0
®
from Illumina®. The MicroPlex kit uses a 3-step protocol with
a
single
purification
at
the
end
of the
procedure.IgGThe
H3K4me3
H3K4me3
IgG
IgG
H3K27ac
H3K27ac
IgG Illumina
workflow for library preparation is composed of 5 steps with several purification procedures.
www.diagenode.com
22
00
IgG
IgG
H3K27ac
H3K27ac
IgG
IgG
ChIP
ChIPH3K27me3
H3K27me3
1818
Sat2
Sat2
ZnF510
ZnF510
cfos
cfos
GAPDH
GAPDH
TSS
TSS
2020
1010
2 2
44
2525
6 6
2 2
66
H3K4me3
H3K4me3
1515
Myt1
Myt1
TSH2B
TSH2B
GAPDHp
GAPDHp
EIF4A2p
EIF4A2p
1616
1414
1212
6 6
4 4
5 5
2 2
TM
0 0
0 0
H3K9me3
H3K9me3
IgG
IgG
25
25
20
20
ChIPH3K27me3
H3K27me3
ChIP
18
18
Sat2
Sat2
ZnF510
ZnF510
cfos
cfos
GAPDHTSS
TSS
GAPDH
16
16
14
14
Figure 4. ChIP efficiency on 10,000 cells.
00
The qPCR was
performedIgG
with primers for two
H3K9me3
IgG
H3K9me3
positive loci and two negative loci for each ChIP
assay. Figures show the recovery, expressed
as a percentage of input (the relative amount
of immunoprecipitated DNA compared to input
DNA after qPCR analysis).
Myt1
Myt1
TSH2B
TSH2B
GAPDHp
GAPDHp
EIF4A2p
EIF4A2p
2%
12
12
00
Matched by Broad
Institute dataset
Unmatched by Broad
Institute dataset
A. The 36 bp tags were mapped to the
human genome with the ELAND aligner.
During the subsequent peak calling by
SICER the enrichments from low cell
numbers could be identified with as
much confidence as from millions of
cells.
B: The datasets were analyzed and
compared with each other and to the
reference data generated by the Broad
Institute. We proved that our low cell
samples are consistent and have very
high similarity, and even the 30 pg
sample fulfils the Encode criteria (min.
80% of the top 40% of the peaks should
overlap.)
Conclusions
True MicroChIP Kit:
•The first and unique ChIP kit for very low starting cell numbers
•Optimized
for an automated format
H3K27me3
IgG
H3K27me3
IgG
•Fully validated using ChIP-qPCR on multiple key epigenetic marks
•ChIP-seq validation using ChIP’d DNA from as low as 10,000 cells with the MicroPlex kit without preamplification
MicroPlex Library Preparation Kit:
•Fast and simplified protocol for NGS library preparation
•Efficient library preparation on picogram amount of DNA without pre-amplification
•Multiplexing capacity of up to 12 samples using standard Illumina® index tags
•Compatible with all Illumina® sequencing platforms
1010
8 8
1010
ChIPH3K9me3
H3K9me3
ChIP
15
15
ChIP assays
were performed on 10,000 Hela 1010
cells with several Diagenode antibodies: 88
10
10
H3K4me3
antibody (0.25 µg/reaction), H3K27ac 6
6
(0.1µg/reaction), H3K9me3 (0.5 µg/reaction) and 4
4
55 (0.25 µg/reaction). Identical quantity
H3K27me3
22
of IgG was used as a control.
88
ChIP
ChIPH3K9me3
H3K9me3
• 1 μg DNA input recommended
•16
13 user-supplied reagent
16
EIF4A2p
EIF4A2p
•> 50 pipetting steps
ACTBp
ACTBp
•14
8 sample transfers Myt1
14
Myt1
• 5 purifications
TSH2B
TSH2B
12
•12
1 clean-up at end
d.
1010
%input
%input
a.
10
10
22
NGS-READY DNA
11
hours
ChIP
ChIP
H3K27ac
H3K27ac
EIF4A2
EIF4A2
prom
prom
GAPDH
GAPDH
prom
prom
MBMB
ex2ex2
Sat2
Sat2
12
12
44
Amplify
Clean-up
%input
%input
a.
•
•
•
•
•
•
88
EIF4A2p
EIF4A2p
ACTBp
ACTBp
Myt1
Myt1
TSH2B
TSH2B
14
14
66
%input
%input
(2)
10
10
%input
%input
Amplify
Clean-up
16
16
EIF4A2prom
prom
EIF4A2
GAPDH
prom
GAPDH prom
MB
ex2
MB ex2
Sat2
Sat2
12
12
(1)
ChIPH3K27ac
H3K27ac
ChIP
%input
%input
Illumina® TruSeqTM DNA LS Prep Kit
%input
%input
Bioruptor ® Pico
MicroPlex
%input
%input
iDeal ChIP-seq kit
2. Proven and reliable ChIP results with the True MicroChIP Kit using Diagenode’s Premium
ChIP-seq
%input
%input
True MicroChIP
ChIP assays were performed on 10,000
Hela cells with the Diagenode antibody
H3K4me3 (0.25 µg/reaction) on the IPStar Compact. 0.25 µg of IgG was used
as a control. The qPCR was performed
with primers for the positive loci EIF4A2
promoter and GAPDH TSS and the
negative loci Myoglobin exon 2 and Sat2.
Figure shows the recovery, expressed
as a percent of input (the relative amount
of immunoprecipitated DNA compared
to input DNA after qPCR analysis).
IgG
High Fidelity Amplification
Primer A
Figure 5. Automation of ChIP assay on
10,000 cells.
H3K27me3
H3K27me3
IgG
IgG
MicroPlex Library Preparation kit x12 contains ThruPLEX technology developed and manufactured by Rubicon Genomics, Inc.,Ann Arbor,
Michigan, USA and covered by US Patent 7,803,550; EP1924704; and US and international patents pending.
The development of the True MicroChIP kit has partly been funded by FP-7 collaborative project (N°221952)
For more information, please contact: for Europe, [email protected] & for the US and Canada, contact [email protected]
PO-µChIP-µPLEX-A3-V1_21_01_13
Abstract
3. True MicroChIP kit compatible with Automated System IP-Star® Compact