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

ChIP was performed as described before [29]. Briefly, the MAGnify™ Chromatin Immunoprecipitation System (Life Technologies, 49–2024) was used with some modifications. Sonication was performed using the Biorupter UCD300 (Diagenode) to obtain chromatin fragments of approximately 100–300 bp. The following antibodies were used for ChIP: anti-H3K4me2 (Abcam ab7766), anti-H3K4me3 (Abcam ab8580), and anti-MyoD (Santa Cruz, sc-760). Sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit (Bio Scientific, 5143) according to an in-house modified protocol. The libraries were 51 bp single-end sequenced on an Illumina HiSeq 2000 platform. Base calling was performed with the Illumina Casava pipeline version 1.8.0. Initial sequencing quality assessment was based on data passing the Illumina Chastity filter.

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