Histone H3K9me3 ChIP-Seq in 5-FU-Resistant Cells

Native chromatin was prepared from LS411N-5FU-R cells and digested with micrococcal nuclease to generate mononucleosomes using the Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling) according to the manufacturer’s instruction. Chromatin fragments were then incubated with anti-H3K9me3 antibody (Abcam) and the chromatin-antibody complexes were isolated using Protein A agarose and salmon sperm DNA (Millipore) according to the manufacturer’s instructions. The ChIP DNA fragments were then prepared using the TruSeq ChIP Sample Preparation Kit (Illumina) and sequenced using High-Seq 2500. Sequence reads in the FASTQ format were generated by the Illumina pipeline CASAVA 1.8.2, and were analyzed using MACS 1.4.2 as described (36 (link)). Peaks were annotated using in-house scripts and genes that have a peak in a promoter were fed into Panther to perform functional analysis.

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Paschall A.V., Yang D., Lu C., Choi J.H., Li X., Liu F., Figueroa M., Oberlies N.H., Pearce C., Bollag W.B., Nayak-Kapoor A, & Liu K. (2015). H3K9 Trimethylation Silences Fas Expression to Confer Colon Carcinoma Immune Escape and 5-Fluorouracil Chemoresistance. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1868-1882.

Publication 2015

Simple ChIP Enzymatic Chromatin IP Kit anti-H3K9me3 antibody Protein A agarose salmon sperm DNA TruSeq ChIP Sample Preparation Kit CASAVA 1.8.2

Agarose Anti antibody Antibody Cells Chip Chromatin Enzymatic Genes Macs 1 Micrococcal nuclease Protein a Salmon Sperm

Corresponding Organization : Zhejiang University

Other organizations : University of North Carolina at Greensboro, Mycosynthetix (United States)

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Variable analysis

independent variables

Preparation of native chromatin from LS411N-5FU-R cells
Digestion of chromatin with micrococcal nuclease to generate mononucleosomes

dependent variables

Enrichment of H3K9me3-associated chromatin fragments
Sequencing of ChIP DNA fragments

control variables

Use of the Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling)
Use of anti-H3K9me3 antibody (Abcam)
Use of Protein A agarose and salmon sperm DNA (Millipore)
Use of the TruSeq ChIP Sample Preparation Kit (Illumina)
Sequencing using High-Seq 2500
Data analysis using MACS 1.4.2 and in-house scripts

positive controls

None specified

negative controls

None specified

Annotations

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