Chromatin Regulation Protocols in Cell Lines

Eco-Pack 2–293 cells (Clontech) and 293T cells (gifted from Dr. Yao [Ito et al., 2001 (link)]) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum. Wild-type and Suv39h dn iMEFs (Lachner et al., 2001 (link)) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 0.1 mM beta-mercaptoethanol, and 1x non-essential amino acids. The following antibodies were used in this study: anti-FLAG-M2 (F3165: Sigma-Aldrich, RRID:AB_259529), anti-FLAG-M2-HRP (A8592: Sigma-Aldrich, RRID: AB_439702), anti-α-tubulin (T5168: Sigma, RRID: AB_477579), anti-Suv39h1 (8729: Cell Signaling, RRID: AB_10829612), anti-H3 (ab21054: Abcam, RRID: AB_880437), anti-H3K9me3 (ab8898: Abcam, RRID: AB_306848 and 2F3 (RRID: AB_2616099)(Chandra et al., 2012 (link)), anti-HP1β (BMP002: MBL, RRID: AB_843158), and anti-GST (27-4577-01: GE Healthcare, RRID: AB_771432).

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Shirai A., Kawaguchi T., Shimojo H., Muramatsu D., Ishida-Yonetani M., Nishimura Y., Kimura H., Nakayama J.I, & Shinkai Y. (2017). Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly. eLife, 6, e25317.

Publication 2017

Eco-Pack 2–293 cells F3165 anti-FLAG-M2-HRP A8592 anti-α-tubulin T5168 ab21054 ab8898

293t cells Antibodies Cells Eagle Essential amino acids Fetal calf serum Mercaptoethanol α tubulin

Corresponding Organization :

Other organizations : RIKEN, National Institute for Basic Biology, Nagoya City University, Yokohama City University, RIKEN Center for Biosystems Dynamics Research, Tokyo Institute of Technology

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Variable analysis

independent variables

Cell lines: Eco-Pack 2–293 cells, 293T cells, wild-type iMEFs, and Suv39h dn iMEFs

dependent variables

Not explicitly mentioned

control variables

Cell culture media: Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, 0.1 mM beta-mercaptoethanol, and 1x non-essential amino acids
Antibodies used: anti-FLAG-M2, anti-FLAG-M2-HRP, anti-α-tubulin, anti-Suv39h1, anti-H3, anti-H3K9me3, anti-HP1β, and anti-GST

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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