Library preparation for ChIP-seq

How to choose your library preparation kit?

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.