Chromatin Shearing: A Method to Generate Chromatin Fragments Using Enzymatic Digestion

Abstract

Source: Fuchs, A. R. et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp.(2018).

This video describes the protocol for chromatin shearing using micrococcal nuclease enzymatic digestion. Chromatin shearing is an essential step in Chromatin immunoprecipitation or ChIp workflow, which helps investigate protein-DNA interaction in human chronic myeloid leukemia cells. In the feature protocol chromatin shearing is performed using sonication.

Protocol

1. Fixation, Cell Lysis, and Chromatin Shearing NOTE: Patient samples and Jurkat cells are fixed and lysed with a commercially available ChIP kit according to the manufacturer's instructions with modifications as described previously. The fixation is performed under a laminar flow hood. Prepare a 1.5 mL tube with 1 mL of 37% formaldehyde, a 1.5 mL tube with 1 mL of 1.25 M glycine, a 5 mL tube with 4 mL of 1x PBS and a box with ice for the fixat…

Representative Results

Figure 1: Examples of antibody performance in fixed Jurkat cells assessed by SDS-PAGE and Western-Blot. (a) Jurkat cells were fixed for 0 min (bands A and D), 2.5 min (bands B and E), or 5 min (C and F) and the αNFAT2-antibody (clone 7A6) from different manufacturers (bands A-C = manufacturer 1, bands D-F = manufacturer 2) was compared. The antibody o…

Materials

37 % Formaldehyde p.a., ACS	Roth	4979.1
1 X PBS	Sigma Aldrich	D8537
1.5 mL tube shaker Themomixer comfort	Eppendorf	5355 000.011	Can be substituted with similar instruments
M220 Focused-ultrasonicator	Covaris	500295
20X Bolt MES SDS Running Buffer	Thermo Scientific	B0002
Tris Buffered Saline (TBS-10X)	Cell Signaling	#12498
iBlot 2 Gel Transfer Device	Thermo Scientific	IB21001
iBlot 2 Transfer Stacks, nitrocellulose, regular size	Thermo Scientific	B23001
big Centrifuge	Eppendorf	5804R	Can be substituted with similar instruments
DNA LoBind Tube 1.5 mL	eppendorf	22431021
Halt Protease and Phosphatase Inhibitor Cocktail (100X)	Thermo Scientific	78440
Small Centrifuge	Thermo Scientific	Heraeus Fresco 17
Density gradient medium Biocoll (Density 1,077 g/ml)	Merck	L 6115