Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

Abstract

Source: Dong, X., et.al., Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFRα+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis. J. Vis. Exp. (2018)

In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Purification of PDGFRα Positive Oligodendrocyte Lineage Cells from Mouse Brain (modified from previously described immunopanning protocols)

1. Preparation of an immunopanning plate for PDGFRα positive cell selection and 2 plates for the depletion of endothelial cells and microglia.
   NOTE: Please note that Petri dishes but not cell culture dishes work for immunopanning experiment; if the purified cells will be used for cu....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/ Equipment
Banderiaea simplicifolia lectin 1	Vector Laboratories	# L-1100
Rat anti-PDGFRa antibody	BD Bioscience	# 558774
Neural tissue dissociation Kit (P)	MACS Miltenyi Biotec	# 130-092-628
Accutase	STEMCELL technologies	# 07920
TRIzol	Thermo Fisher	# 15596026
Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody	Millipore	# AB5320
Bioruptor Pico sonication device	Diagenode	# B01060001
True MicroChIP kit	Diagenode	# C01010130
Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)	Thermo Fisher	# 15593031
NucleoSpin Gel and PCR Clean-Up kit	MACHEREY-NAGEL	# 740609
Quant-iT PicoGreen dsDNA Assay Kit	Thermo Fisher	# P11496
DNA SMART ChIP-Seq kit	Clontech Laboratories	# 634865
Agencourt AMPure XP	Beckman Voulter	# A63880
GlycoBlue	Thermo Fisher	# AM9516
Pico-green	Thermo Fisher	# P11496
D-PBS	Thermo Fisher	# 14190-144
SYBR Green master mix	Bio-Rad Laboratories	# 1725124
DMEM/F12	Fisher Scientific	# 11-320-033
Penicillin-Streptomycin (P/S)	Fisher Scientific	# 15140122
N-2 Supplement	Fisher Scientific	# 17502048
B-27 Supplement	Fisher Scientific	# 17504044
Insulin	Sigma	# I6634	Prepare 0.5 mg/ml insulin solution by dissolving 5 mg insulin in 10 ml water and 50 μl of 1 N HCl.
Bovine Serum Albumin, suitable for cell culture (BSA)	Sigma	# A4161	Prepare 4% BSA solution by dissolving 4 g BSA in 100 ml D-PBS and adjust the pH to 7.4
Fibroblast Growth Factor basic Protein, Human recombinant (bFGF)	EMD Millipore	# GF003
HUMAN PDGF-AA	VWR	# 102061-188
Poly-D-lysine	VWR	# IC15017510	Prepare 1 mg/ml poly-D-lysine solution by dissolving 10 mg poly-D-lysine in 10 ml water and dilute 100 times when using.
Falcon Disposable Petri Dishes, Sterile, Corning, 100x15mm	VWR	# 25373-100
Falcon Disposable Petri Dishes, Sterile, Corning, 150x15mm	VWR	# 25373-187
HBSS, 10X, no Calcium, no Magnesium, no Phenol Red	Fisher Scientific	# 14185-052
Trypan Blue	Stemcell Technologies	# 07050
Protease inhibitor cocktail	Sigma	# 11697498001
Primer names used for qPCR			Primer sequences used for qPCR
Mbp-F:			CTATAAATCGGCTCACAAGG
Mbp-R:			AGGCGGTTATATTAAGAAGC
Iba1-F:			ACTGCCAGCCTAAGACAACC
Iba1-R:			GCTTTTCCTCCCTGCAAATCC
Mog-F:			GGCTTCTTGGAGGAAGGGAC
Mog-R:			TGAATTGTCCTGCATAGCTGC
GAPDH-F			ATGACATCAAGAAGGTGGTG
GAPDH-R			CATACCAGGAAATGAGCTTG
Tuj1-F			TTTTCGTCTCTAGCCGCGTG
Tuj1-R			GATGACCTCCCAGAACTTGGC
PDGFRα-F			AGAGTTACACGTTTGAGCTGTC
PDGFRα-R			GTCCCTCCACGGTACTCCT