Illustra GFX PCR DNA and Gel Band Purification kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.

Publication protocol

Monocyte isolation
A blood sample of 30 ml was taken and peripheral blood mononuclear cells were isolated using ficoll-based density gradient centrifugation. Further isolation of CD14++ CD16- monocytes was conducted using magnetic cell sorting to deplete CD16++ monocytes and separate CD14++ monocytes. Purity of isolation was checked by flow cytometric analysis of the CD14+ cell fraction.

Cell preparation
Cells were cross-linked for 10 min using formaldehyde (final concentration 1%).

ChIP protocol
The ChIP protocol was optimized for small amounts of monocytes. Chromatin has to be sheared using ultrasound sonication. To gain satisfying results, a specialised sonication programme with additional adapted cooling is indispensable. We designed a shortened ChIP protocol, where immunoprecipitation, reverse cross-linking and DNA purification is executed within 2 days.

Library amplification
Library amplification was conducted using a specially modified library preparation kit protocol to supervise library amplification with the presence of SYBR Green in a real-time PCR machine.

ChIP sequencing
Sequencing of the libraries was performed on an Illumina HiSeq 2000.

Computational data analysis
In addition to identification of enriched and depleted genomic regions and annotations, correlation analysis was performed to compare and validate data quality. Furthermore we compared our data to publicly available expression data in order to identify functional relationships between chromatin states and gene expression.



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Manufacturer protocol

Download the product protocol from Fisher Scientific for Illustra GFX PCR DNA and Gel Band Purification kit below.

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