Chromatin preparation guidelines and tips

The epigenetic services team at Active Motif has been evolving & optimizing our ChIP chromatin preparation protocols for 10 years. Most sample types are amenable to a standard chromatin preparation protocol but others can be difficult and require a specialized approach.

Below are tips and information about chromatin preparation for various sample types.

Cell lines
Cell Plates

Starting amount: 10 million cells
 Protocol: For most cell lines one 15 cm plate will yield enough chromatin for multiple ChIP reactions.

Mouse tissues
Mouse

Starting amount: 100 to 200 mg as a starting point. Yield will vary between different tissue types.
Protocol: Specialized protocols for fixation and chromatin preparation.
Notes: Requires tissue dissociation using a hand held homogenizer.

Active Motif has performed ChIP-Seq from >25 different mouse tissues

Mouse tissues used for chromatin preparation
     Atria      Brown adipose      Cerebellum      Colon
     Diaphragm      Face      Heart      Cerebellum
     Hindbrain      Hippocampus      Kidney      White adipose
     Lung      Mammary      Muscle      Pancreas
     Pituitary      Retina      Thymus      Spinal cord
     Spleen      Stomach      Testicle      Sciatic nerve
     Tooth bud      Uterus      Liver      Whole brain

Human biopsies and postmortem tissue
Musculoskeletal

Starting amount: 100 to 200 mg as a starting point. Yield will vary between different tissue types.
 Protocol: Specialized protocols for fixation and chromatin preparation.
Notes: The protocol is the same as that used for mouse tissues. This protocol works with all solid tumors and postmortem tissues.

T cells & B cells
T Cells

Starting amount: >20 million cells
Protocol: Specialized protocols for fixation and chromatin preparation.
Notes: Requires 2X sonication compared to a standard preparation. Cell loss can occur during wash steps after fixation. Yields will be much lower than cell line preparations using comparable cell numbers.

Mammary tissue & Adipose cells
Adipose Tissue

Starting amount: >500mg
 Protocol: Specialized protocol for cells containing fat.
 Notes: Fat content creates layers after spinning lysed cells and must be separated in order to isolate the fat cells. More starting material is required than that used for other tissue types.

Oligodendrocytes and neurons
Oligodendrocytes

Starting amount: >5 million cells
 Protocol: Specialized protocol that is similar to our T and B cell protocol.

Drosophila
Drosophila

Starting amount: Varies depending on the tissue type.
 Protocol: Specialized protocol for Drosophila.
 Notes: Trichomes, bristles and other structures are not lysed in the protocol and must be removed during chromatin preparation.

Yeast
Yeast Cells

Starting amount: 100µl cell pellet
 Protocol: Specialized fixation and chromatin preparation.
 Notes: Yeast cell wall requires a BeadBeater for cell lysis.

Plant Tissue
Plant Cells

Starting amount: Varies depending on the species and tissue type.
 Protocol: Specialized fixation and chromatin preparation.
 Notes: Fixation must occur under vacuum, then freezing with liquid nitrogen followed by grinding the sample into a powder to facilitate chromatin extraction.