ChIP Rat - PCCL3

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

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HighCell# ChIP kit

Diagenode

Upstream tips	Protocol tips	Downstream tips
After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.	-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. -Chromatin from 1,000 cells is sufficient for one ChIP assay.	-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Upstream tips
After cell harvesting, chromatin must be sheared to 200 to 600 bp before ChIP. -Optimize shearing conditions for your specific cell type and fixation protocol before starting a ChIP. Therefore, start with a small sample (1x 10e5 to 1x 10e6 cells) and check the shearing efficiency.
Protocol tips
-Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility. -Make sure that there are no crystals in the Buffer B. Gently heat and mix until crystals disappear. -Chromatin from 1,000 cells is sufficient for one ChIP assay.
Downstream tips
-For longer term storage, aliquot 130 µl of sheared chromatin into cryotubes, snap-freeze in liquid nitrogen, and store at -80°C.

Manufacturer protocol Publication protocol 1 Relevant paper

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