yFS661 was synchronized at G1, formaldehyde cross linked and subjected to MNase ChIP with rabbit anti-Mcm2-7 polyclonal antibody (UM185, gift from Steve Bell) (Bowers et al. 2004 (link)) and protein G agarose (Sigma) as described (Liu et al. 2005 (link)). Briefly, 5 × 109 G1-arrested cells were cross linked with 0.1% formaldehyde for 15 min and quenched with 125 mM glycine. MNase-digested chromatin was prepared by spheroplasting cells with Zymolyase 100T (Seikagaku), recovering chromatin by centrifugation, and treating with MNase (Worthington) to digest it to mononucleosomal and sub-mononucleosomal fragments.
Protocol detail
ChIP-seq protocol for Mcm2-7 proteins
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Agarose
Anti antibody
Cells
Centrifugation
Chip
Chromatin
Formaldehyde
Glycine
Mcm2
Protein g
Rabbit
Zymolyase
Corresponding Organization :
Other organizations : University of Massachusetts Chan Medical School, Simon Fraser University
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Variable analysis
independent variables
- Formaldehyde cross-linking
- MNase treatment
- Enrichment of Mcm2-7 proteins on chromatin
- G1-arrested cells
- Rabbit anti-Mcm2-7 polyclonal antibody (UM185)
- Protein G agarose
- Positive control: Rabbit anti-Mcm2-7 polyclonal antibody (UM185)
- No negative control was explicitly mentioned.
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