Chromatin Immunoprecipitation (ChIP) Protocol

ChIP assays were performed as described [33 (link),34 (link)]. Briefly, 2.5 million cells were crosslinked with 37% w/v formaldehyde, quenched with 2.5M glycine, washed 3 times with ice cold PBS, and sonicated in a Bioruptor (Diagenode). The following primary antibodies were utilized for incubation of crosslinked chromatin overnight: HDAC1 (Abcam #31263, 3μg), HDAC2 (Abcam #12169, 3μg) pan-H4ac (Upstate #06–866, 5μg), H3K9Ac (Abcam #12179, 5μg), H3K9me3 (Abcam #8898, 5μg), SIN3A (Abcam #3479, 5μg) and as a negative control IgG Rabbit (Cell Signaling Technologies #2729S, 3 or 5μg), IgG Rabbit (Santa Cruz #2027, 3 or 5μg), or IgG Mouse (Santa Cruz #2025, 3 or 5μg,). Protein A/G magnetic beads (Pierce #88802) were added at 50 μL per ChIP sample. ChIP-qPCR primers were designed using Primer3 web tool and are listed in S5 Table.

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Somanath P., Herndon Klein R, & Knoepfler P.S. (2017). CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells. PLoS ONE, 12(10), e0185627.

Publication 2017

Bioruptor HDAC2 H3K9Ac H3K9me3 IgG Rabbit IgG Mouse

Antibodies Cells Chip Chip assays Chromatin Cold Formaldehyde Glycine Mouse Primers Protein a Protein g Rabbit

Corresponding Organization : University of California, Davis

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Variable analysis

independent variables

37% w/v formaldehyde crosslinking
2.5M glycine quenching
Sonication in Bioruptor
Primary antibodies: HDAC1, HDAC2, pan-H4ac, H3K9Ac, H3K9me3, SIN3A

dependent variables

Enrichment of HDAC1, HDAC2, pan-H4ac, H3K9Ac, H3K9me3, SIN3A at target genomic regions

control variables

Number of cells used (2.5 million)
Washing with ice cold PBS

controls

Positive controls: None specified
Negative controls: IgG Rabbit (Cell Signaling Technologies #2729S, 3 or 5μg), IgG Rabbit (Santa Cruz #2027, 3 or 5μg), IgG Mouse (Santa Cruz #2025, 3 or 5μg)

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