Diagenode

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NGS Library preparation

Library preparation for ChIP-seq

Library preparation following ChIP can be challenging due to the limited amount of DNA recovered after ChIP. Diagenode has developed the optimal solutions for ChIP-seq using two different approaches: the ligation-based library preparation on purified DNA or the tagmentation-based ChIPmentation.

  • Standard input library prep

    The iDeal Library Preparation Kit reliably converts DNA into indexed libraries for next-generation sequencing, with input amounts down to 5 ng. Our kit offers a simple and fast workflow, high yields, and ready-to-sequence DNA on the Illumina platform.

    Features

    • Sample: Fragmented dsDNA
    • Input: 5 ng – 1 µg
    • Fast protocol: 3 hours
    • Easy processing: 3 steps
    • Indexing: single indexes for multiplexing up to 24 samples
    • Manual and automated protocols available
    • Sequencing technology: Illumina

    Applications

    • MeDIP-seq library prep
    • Genomic DNA sequencing
    • High input ChIP-seq

The TAG Kit for ChIPmentation offers an optimized ChIP-seq library preparation solution based on tagmentation. This kit includes reagents for tagmentation-based library preparation integrated in the IP and is compatible with any ChIP protocol based on magnetic beads. The primer indexes for multiplexing must be purchased separately and are available as a reference: 24 SI for ChIPmentation, Cat. No. C01011031. Alternatively, for histone marks, Diagenode proposes the complete solution (including all buffers for ChIP, tagmentation and multiplexing): ChIPmentation for Histones and µChIPmentation for histones.

Features

  • Sample: chromatin-antibody-magnetic beads complexes
  • Input: chromatin from 5 K – 4 M cells
  • Easy and fast protocol
  • Compatible with any ChIP protocol based on magnetic beads
  • No adapter dimers
  • Sequencing technology: Illumina

Applications

TAG kit for ChIPmentation

  • ChIPmentation library preparation

24 SI for for ChIPmentation

  • ChIPmentation library preparation
  • Tagmentation-based library preparation methods like ATAC-seq, CUT&Tag

KITS

Cat. No. Product Format
C01011030 TAG Kit for ChIPmentation 24 rxns
C01011031 24 SI for ChIPmentation 24 rxns

How to choose your library preparation kit?

Sample

Chromatin-antibody-beads complex

Purified DNA

Purified DNA

Application

ChIPmentation

ChIP-seq library prep
Low input DNA sequencing

MeDIP-seq library prep
Genomic DNA sequencing
High input ChIP-seq

Input

Chromatin: 5 K to 4 M cells

DNA: 50 pg – 50 ng

DNA: 5 ng – 1 µg

Multiplexing

Up to 24 samples

Up to 384 samples

Up to 48 samples

Up to 24 samples

Indexes

Single indexes (SI)

Dual indexes (DI)

Single indexes (SI)

Single indexes (SI)

Kit

TAG Kit for ChIPmentation
(indexes not included in the kit)

Kit
C01011030 – 24 rxns

Single indexes
C01011031 – 24 SI/24 rxns

MicroPlex Library Preparation Kit v3
(dual indexes not included in the kit)

Kit
C05010001 - 48 rxns
C05010002 - 96 rxns


Unique dual indexes
C05010008 - Set I 24 UDI / 48 rxns
C05010009 - Set II 24 UDI/ 48 rxns

Dual indexes
C05010003 - 24 DI/ 48 rxns
C05010004 - Set I 96 DI/ 96 rxns
C05010005 - Set II 96 DI/ 96 rxns
C05010006 - Set III 96 DI/ 96 rxns
C05010007 - Set IV 96 DI/ 96 rxns

MicroPlex Library Preparation Kit v2
(single indexes included in the kit)

C05010012 - 12 SI/ 12 rxns
C05010013 - 12 SI/ 48 rxns

iDeal Library Preparation Kit
(Set 1 of indexes included in the kit)

C05010020 - 12 SI/ 24 rxns

Index Primer Set 2

C05010021 - 12 SI/ 24 rxns

Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has become the gold standard to investigate genome-wide epigenetic profiles. However, ChIP from a limited amount of cells has been a challenge. Here we provide a complete and robust workflow solution for successful ChIP-seq from small numbers of cells using the True MicroChIP kit and MicroPlex Library Preparation kit.

APPLICATION NOTE

ChIP efficiency on 10,000 cells

From minuscule amounts to magnificent results:
reliable ChIP-seq data from 10,000 cells with the True MicroChIP™ and the MicroPlex Library Preparation™ kits.

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APPLICATION NOTE

Quality control check of a ChIP-seq library on the Fragment Analyzer. High Efficiency ChIP performed on 10,000 cells

Best Workflow Practices for ChIP-seq Analysis with Small Samples

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TAG Kit for ChIPmentation

  1. What is the difference between tagmentation and ChIPmentation?
    Tagmentation is a reaction where an enzyme (a transposase) cleaves DNA and incorporates sequencing adaptors at the ends of the fragments in one step. In our ChIPmentation technology we combine chromatin immunoprecipitation and tagmentation in one streamlined workflow where the tagmentation step occurs directly on chromatin.

  2. What is the expected concentration of ChIPmentation libraries?
    The concentration of libraries that you need to reach will depend on the sensitivity of the machine and kits that you will use to perform the quality control and the sequencing of your libraries. Usually a concentration of 4-8 ng/μl is enough for a quality control using the Qubit High Sensitivity assay (ThermoFischer Scientific) and the High Sensitivity chip for BioAnalyzer (Agilent) and for sequencing on Illumina HiSeq3000/4000.

  3. Does the ChIPmentation approach work on plants?
    Our ChIPmentation solution has been validated on human cells and we do not have any data on plants. It should be compatible. We would recommend using our Universal Plant ChIP Kit in combination with the TAG Kit for ChIPmentation and the 24 SI for ChIPmentation.

  4. What is the size of the fragments after the tagmentation?
    The size of the fragments at the end of the ChIPmentation protocol can vary depending on many parameters like the shearing efficiency, the antibody used or the tagmentation time. However, with our standard protocol we usually obtain a library peak which is around 200-300 bp (see example of results at the end of the manual). If many fragments larger than 500 bp are present , the best would be to contact your sequencing provider to ask what their requirements are, because it can vary depending on the sequencer. If you want to remove the large fragments you can use the size selection protocol described in the manual.

  5. What is the size of the adapters?
    The sum of the adapters is 128 bp.

MicroPlex Library Preparation Kit

  1. Can I use the available Illumina primers and validate them with the MicroPlex Kit v2?
    Although the final flanking sequences of MicroPlex are the same as those used by Illumina, the PCR primers are not identical and part of them is supplied with the buffer. For this reason Illumina primers will not work as substitute.

  2. The BioAnalyzer profile of purified library shows the presence of low molecular weight peaks (primers/adaptors) in the samples. Should I re- purify the samples or they can be used directly to the sequencing? If the second purification is recommended, which ratio sample/AMPure beads should I use?
    You can do a second round of purification using 1:1 ratio of AMPure beads to sample and this should get rid of the majority of the dimers.

  3. I am going to use the MicroPlex Library Preparation Kit v2 on ChIP samples . Our thermocycler has ramp rate 1.5°/s max while the protocol recommends using a ramp rate 3 to 5°/s. How would this affect the library prep?
    We have not used a thermocycler with a ramp rate of 1.5 °C, which seems faster than most of thermocyclers. Too fast of a ramp rate may affect the primer annealing and ligation steps.

  4. What is the function of the replication stop site in the adapter loops?
    The replication stop site in the adaptor loops function to stop the polymerase from continuing to copy the rest of the stem loop.

  5. I want to do ChIP-seq. Which ChIP-seq kit can I use for sample preparation prior to Microplex Library Preparation Kit v2?
    In our portfolio there are several ChIP-seq kits compatible with Microplex Library Preparation Kit v2. Depending on your sample type and target studied you can use the following kits: iDeal ChIP-seq Kit for Transcription Factors (Cat. No. C01010055), iDeal ChIP-seq Kit for Histones (Cat. No. C01010051), True MicroChIP kit (Cat. No. C01010130), Universal Plant ChIP-seq Kit (Cat. No. C01010152). All these kits exist in manual and automated versions.

  6. Is Microplex Library Preparation Kit v2 compatible with exome enrichment methods?
    Microplex Library Preparation Kit v2 is compatible with major exome and target enrichment products, including Agilent SureSelect®, Roche NimbleGen® SeqCap® EZ and custom panels.

  7. What is the nick that is mentioned in the kit method overview?
    The nick is simply a gap between a stem adaptor and 3’ DNA end, as shown on the schema in the kit method overview.

  8. Are the indexes of the MicroPlex library preparation kit v2 located at i5 or i7?
    The libraries generated with the MicroPlex kit v2 contain indices located at i7.

  9. Is there a need to use custom index read primers for the sequencing to read the 8nt iPCRtags?
    There is no need for using custom Sequencing primer to sequence MicroPlex libraires. MicroPlex libraries can be sequenced using standard Illumina Sequencing kits and protocols.

  10. What is the advantage of using stem-loop adapter in the MicroPlex kit?
    There are several advantages of using stem-loop adaptors. First of all, stem-loop adaptors prevent from self-ligation thus increases the ligation efficiency between the adapter and DNA fragment. Moreover, the background is reduced using ds adaptors with no single-stranded tails. Finally, adaptor-adaptor ligation is reduced using blocked 5’ ends.

IDeal Library Preparation Kit

  1. Are the index from the iDeal library Prep kit compatible with the MicroPlex library prep kit?
    No, it is important to use only the indexes provided in the MicroPlex kit to ensure proper library preparation with this kit

Products

Cat. No.ProductFormatPrice
C05010003 24 Dual indexes for MicroPlex Kit v3 /48 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq library prep from ChIP-derived DNA. The kit MicroPlex v3...
48 rxns $380.00
C05010004 96 Dual indexes for MicroPlex Kit v3 – Set I /96 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq library prep from ChIP-derived DNA. The kit MicroPlex v3...
96 rxns $1,155.00
C05010005 96 Dual indexes for MicroPlex Kit v3 – Set II /96 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq library prep from ChIP-derived DNA. The kit MicroPlex v3...
96 rxns $1,155.00
C05010006 96 Dual indexes for MicroPlex Kit v3 – Set III /96 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq library prep from ChIP-derived DNA. The kit MicroPlex v3...
96 rxns $1,155.00
C05010007 96 Dual indexes for MicroPlex Kit v3 – Set IV /96 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq library prep from ChIP-derived DNA. The kit MicroPlex v3...
96 rxns $1,155.00
C05010001 MicroPlex Library Preparation Kit v3 /48 rxns
Diagenode’s MicroPlex Library Preparation Kits v3 have been extensively validated for ChIP-seq samples and are optimized to generate DNA libraries with...
48 rxns $1,400.00
C05010020 iDeal Library Preparation Kit x24 (incl. Index Primer Set 1)
The iDeal Library Preparation Kit reliably converts DNA into indexed libraries for next-generation sequencing, with input amounts down to 5 ng. Our kit offer...
24 rxns $925.00
C05010021 Index Primer Set 2 (iDeal Library Preparation Kit x24)
The Index Primer Set 2 (iDeal Library Preparation Kit x24) contains an additional set of 12  indexes to be used in combination with the iDeal Library Pr...
24 rxns $200.00
C05010012 MicroPlex Library Preparation Kit v2 (12 indexes)
Specifically optimized for ChIP-seqThe MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allow...
12 rxns $800.00

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