This protocol is for doing ChIP from less 100K of cells down to approximately 5K of cells (one neurosphere) note: for histone modification antibodies we have successfully used as little as 500 cells.

Fixation: 1% paraformaldehyde this also acts to crosslink DNA to histones. Use fresh paraformaldehyde, older PAF breaks down and cause side reactions.

If doing neurospheres or Embryoid Bodies collect a single NS or EB in individual eppendorf tubes. rough ratio is 1 neurosphere=1 antibody. Due to the heterogeneity of gene expression in NS or EBs expect to need at least 5 replicates to validate results within an experiment.

NOTE: EBs or NS should also be taken for RT-PCR reactions to be run in parallel.

As a rule, each experiment should be performed on three separate occasions to ensure that the NS or EB differentiation are general features and not the result of experimental error.

For ANY experiment you need at least 2 spheres: 1 for your Antibody and 1 for the IgG/non-specific antibody control.

Reduce volume of media or PBS to a max of 10uls, add 1ul of 10% PAF-this way the whole procedure can be done in one eppendorf tube

Incubate 10 mins @ 37oC (do not increase this time)

PRECLEAR STEP For suspension cells spin down

remove PAF containing media or PBS and add 10uls of ChIP SDS lysis buffer/10K of cells. (for the following all suspension steps are in red-and given assuming 10K of cells)

Add dilution buffer to a total volume of 500uls

Bring total volume to 500uls.

Pre-clear with 20uls of ProteinA/ssDNA slurry for 1hr.

For adherent single colonies

Add 20uls of ChIP SDS lysis buffer (plus protease inhibitors)

Add 100uls of ChIP dilution buffer

Sonicate in bioruptor 12mins (30/30 on/off cycle) [this is the standard ChiP settings] in eppendorf tube. –[we have a diagenode bioruptor, we I have not tried it with a “stick sonicator”]

Bring total volume to 200uls with dilution buffer.

Remove 20uls for INPUT

Antibody incubationSuspensionAdd antibody of interest, incubate overnight @4oC

AdherentSplit into atleast 2 tubes (negative control and Antibody of interest) and a max. of 4 (neg. control plus 3 Antobody of Interests). Incubate o/n.

The antibodies need to be tested, if it is known you can use the ug/ul ration used for larger volume ChIP experiments. This is dependent on quality of antibody. For example for KDM5b we use the same amount of Ab for svChIP as ChIP.

Proceed as usual with ChIP:

1. Precipitate antibody/protein/DNA complex with Protein A beads/ssDNA. Agitate 1-3 hours at 4oC.

2. Wash once with Low salt, incubate 5-15 minutes 3. Wash once with High salt, incubate 5-15 minutes 4. Wash once with LiCl salt, incubate 5-15 minutes 5. Wash twice with TE, incubate 5-15 minutes 6. Elute with 1% SDS in Sodium Bicarbonate buffer (0.1M), 2hours at room temperature.7. Spin briefly, remove supernatant and incubate (supernatant) at 65oC 6 hours no longer than

18 hrs or DNA will breakdown.8. Purify DNA by qiaquick by qiagen. We have found that bead based DNA cleanups have too

low a yield. The best method is old school precipitation methods. Qiagen’s Qiaquick is a good commercial version.

9. Use 10ul of elutant per PCR (for a 20ul reaction)10. PCR genomic locus of interest. We found that probe based methods gave better signal

reproducibility. We successfully used Promega’s plexor system or ABI Taqman probe systems. We have gotten signal using SYBR green but it is variable so we prefer Promega Plexor which is a Fluorescent Primer based system that we can use the same primers as large scale ChIP using SYBR green.

Solutions

Salmon Sperm DNA/Protein A Agarose: 1μg SSDNA/1mLProteinA beads

SDS Lysis Buffer: 1% SDS, 10mM EDTA, 50mM Tris, pH 8.1.

ChIP Dilution Buffer:0.01% SDS, 1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl(pH 8.1), 167mM NaCl.

Low Salt Immune Complex Wash Buffer:0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl(pH 8.1),150mM NaCl.

High Salt Immune Complex Wash Buffer: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl(pH 8.1),500mM NaCl.

LiCl Immune Complex Wash Buffer: 0.25M LiCl,1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris(pH 8.1).

TE Buffer:10mM Tris-HCl, 1mM EDTA(pH 8.0).

If you use this protocol please cite one of the following articles that uses the protocolStalker L and Wynder C. Evaluation of histone modifying enzymes in stem cell populations.-Methods in Molecular Biology. Methods Mol Biol. 2012, 809, 411-26 Corresponding author

Venugopal C, Li N,Wang X, Manoranjan B, Hawkins C, Gunnarsson T, Hollenberg R,Klurfan P, Murty N, Kwiecien J, Farrokhyar F, Provias JP, Wynder C, and Singh SK. Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells. Stem Cell Research (2012) 8, 141–153.

Zhou Q, Dalgard C, Wynder C, and Doughty ML. Valproic acid inhibits neurosphere formation by adultsubventricular cells by a lithium-sensitive mechanism. Neurosci Lett. 2011 Aug 18;500(3):202-6.