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Breast Cancer Regulation
The protocol below is intended for adherent cells. For suspension cells, collect them by centrifugation (start with step stated with a * below).
Note: Do not continue trypsin treatment longer than necessary as prolonged treatment with trypsin may damage the cells. Regularly check if the cells start to detach.
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: The fixation time might require an additional optimization. In general, histone marks require shorter fixation (8 min) than transcriptional factors (10-15 min). Stronger fixation may lead to chromatin resistant to sonication.
Note: If the starting amount of cells was less than 10 million, scale down accordingly (e.g. Use a total of 5 mL lysis buffer C for 5 million cells).
Note: This protocol has been optimized for 1 million cells per ChIP. Although it is possible to use more cells, we recommend performing separate ChIP reactions and pool the samples before purification of the DNA.
Note: 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: Do not disturb the captured beads attached to the tube wall. Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.
Note: Total elution volume for both IP and input samples is 50 µL (1.5 mL tube).
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 (GAPDH SS) and negative (Myoglobin exon 2) control primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade H3K4me3 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 GAIIx and Ion Torrent next-generation sequencers. However, other sequencing systems such as the Illumina HiSeq or the Thermo Fisher SOLiD systems can also be used. Refer to their sequencing protocols for the generation of sequencing data.