MAGnify™ Chromatin Immunoprecipitation System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.	-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Upstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.
Protocol tips
-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Publication protocol

Cardiac tissue was isolated from GD 20 pups in both the nano-TiO2 exposure and control groups. Each litter was considered an n = 1, with cardiac tissue from 5 to 6 pups within each litter being pooled together to collect enough tissue (~25 mg). Chromatin Immunoprecipitation (ChIP) was carried out using the MAGnify™ Chromatin Immunoprecipitation System (Thermo Fisher, Rockford, IL) per manufacturer’s instructions. Briefly, hearts were homogenized and treated with 37% formaldehyde, which was prepared fresh. Cross-linking was stopped with 1.25 M glycine. Samples were pelleted through centrifugation and washed in D-PBS before sonication. Using a Sonicator Ultrasonic Processor XL2015 (Misonix Sonicator, Farmingdale, NY) chromatin was sheared to a size of 500-700 base pairs, determined using gel electrophoresis (Fig. 1a). Chromatin was then isolated through ultracentrifugation (20,000 g) and diluted to ~60 uL of chromatin per immunoprecipitation reaction. Samples from both the control and nano-TiO2 cohorts were incubated with histone 3 lysine 4 tri-methylation (H3K4me3, product number: G.532.8, Thermo Fisher, Rockford, IL) or histone 3 lysine 27 tri-methylation (H3K27me3, product number: G.299.10, Thermo Fisher, Rockford, IL) antibody bound beads. These are two of the most prominently studied and classically applied for activation/repression analysis of gene activity. After incubation, samples were treated to reverse cross-linking solution and Proteinase K to remove bound proteins. DNA was then eluted from beads, using heat, and quantified using a Qubit (Thermo Fisher, Rockford, IL). The TruSeq ChIP Library Preparation Kit (Illumina, Inc., San Diego, CA) was implemented to build the libraries.

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Manufacturer protocol

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