ChIP-seqライブレリー調製

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

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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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