ChIP-qPCR Analysis of Protein-DNA Interactions

ChIP experiments were performed with three independent biological replicates using the approach as described previously (Mishra et al., 2007 (link), 2011 (link)). Antibodies used to capture DNA-protein complexes were anti-HA agarose (A2095; Sigma Aldrich), anti-Myc agarose (A7470; Sigma Aldrich), anti-Flag agarose (A2220; Sigma Aldrich), and anti-histone H3 (ab176842; Abcam). ChIP-qPCR was done using Fast SYBR Green Master Mix in 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA) following conditions described previously (Mishra et al., 2015 (link)). The enrichment as presented as percent input was determined using the _ΔΔC_T method (Livak and Schmittgen, 2001 (link)).

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Mishra P.K., Chakraborty A., Yeh E., Feng W., Bloom K.S, & Basrai M.A. (2021). R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast. Molecular Biology of the Cell, 32(1), 74-89.

Publication 2021

anti-HA agarose anti-Myc agarose anti-Flag agarose ab176842 Fast SYBR Green Master Mix 7500 Fast Real Time PCR System

Agarose Antibodies Biological Chip Histone h3 Protein Sybr green

Corresponding Organization :

Other organizations : National Cancer Institute, National Institutes of Health, Center for Cancer Research, SUNY Upstate Medical University, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Antibodies used to capture DNA-protein complexes
Approach as described previously (Mishra et al., 2007, 2011)

dependent variables

Enrichment as presented as percent input

control variables

Three independent biological replicates
Fast SYBR Green Master Mix in 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA)
Conditions described previously (Mishra et al., 2015)
ΔΔCt method (Livak and Schmittgen, 2001)

positive controls

Anti-histone H3 (ab176842; Abcam)

negative controls

Not explicitly mentioned

Annotations

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