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Breast Cancer Regulation
Note: The fixed cells can be stored at -80°C for up to 4 months. However, we strongly recommend using freshly fixed cells for preparation of sheared chromatin prior to ChIP for ChIP-seq.
Note: This protocol has been optimized for ~4 million cells per ChIP, although it is possible to reduce or increase the amount of cells. For using lower amounts of cells, simply dilute the chromatin in shearing buffer A before adding it to the IP reaction. For higher cell numbers you can increase the cell concentration in the shearing buffer, although this may require an additional optimization of the shearing conditions. Therefore, we recommend performing separate ChIPs and pool the samples before purification of the DNA.
Note: If you will use different amounts of antibody (e.g. when performing a titration curve), we recommend to add the water separately. Also, if required, sodium butyrate NaBu, a potent deacetylase inhibitor, (20 mM final concentration) or other inhibitors can also be added.
Note: Up to two samples can be easily pooled. If more than two samples need to be pooled, process each sample purification individually, pool final eluates at the end of the magnetic bead purification and concentrate.
Note: Following the addition of isopropanol the solution may become cloudy. This is not detrimental to your experiment and will not influence the quality or quantity of your purified DNA.
Note: Keep the beads in liquid suspension during storage at 4°C and at all handling steps, as drying will result in reduced performance.
Note: In order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet, incubate for 30 sec at room temperature on a rotating wheel (40 rpm). Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.
Note: The elution buffer C is suitable for direct qPCR analysis, whole genome amplification, chip hybridization and Next-Generation sequencing.
Before sequencing the samples, we recommend analyzing the immunopreciptated DNA by qPCR using at least one positive and one negative control target. The kit contains a positive (H19 imprinting control region) and negative (Myoglobin exon 2) control primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade CTCF antibody) in SYBR Green qPCR assay using the protocol described below. Use your own method of choice for analyzing the appropriate control targets for your antibodies of interest.
Note: Check carefully supplier’s recommendations about Taq polymerase activation time. These PCR conditions may require optimization depending on the type of Master Mix or qPCR system used.
This protocol has been optimized for ChIP-seq on an Illumina HiSeq sequencer. However, other sequencing systems such as the Illumina MiSeq or the Thermo Fisher SOLiD systems can also be used. Refer to their sequencing protocols for the generation of sequencing data.