Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

Vasso Makrantoni; Daniel Robertson; Adele L. Marston

Sep 22, 2021

Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

DOI

dx.doi.org/10.17504/protocols.io.bn3kmgkw

Vasso Makrantoni¹,
Daniel Robertson¹,
Adele L. Marston¹

¹The Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

Springer Nature Books

Satyavati Kharde

Springer Nature

DOI: dx.doi.org/10.17504/protocols.io.bn3kmgkw

External link: https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10

Collection Citation: Vasso Makrantoni, Daniel Robertson, Adele L. Marston 2021. Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.bn3kmgkw

Manuscript citation:

Makrantoni V., Robertson D., Marston A.L. (2019) Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation. In: Badrinarayanan A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_10

License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it’s working

Created: October 27, 2020

Last Modified: September 22, 2021

Collection Integer ID: 43852

Keywords: Chromatin immunoprecipitation, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Cohesin, Condensin, Mitosis, Meiosis, Scc1, Rec8, Brn1

Abstract

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

Materials

Yeast Strains and Growth Material:
Haploid S. cerevisiae strains of w303 background we have used include: (a) no tag control (AM1176), (b) SCC1-6HA (AM1145), (c) BRN1-6HA (AM5708), (d) SCC2-6HIS-3FLAG (AM6006), and (e) SCC1-6HA pMET3-CDC20 (AM1105) as previously described [9,10,11,12].
For studies of protein occupancy during meiosis we have used diploid S. cerevisiae strains of SK1 background including (a) REC8-3HA ndt80Δ (AM4015), as previously described [13] and (b)REC8-6HIS-3FLAG (AM11000).
Haploid S. pombe strains used for calibration are: (a) RAD21-3HA (spAM76), (b) RAD21-6HA (spAM635), (c) RAD21-6HIS-3FLAG (spAM1863), or (d) CND2-6HA (spAM1862).
YPDA media: 1% yeast extract, 2% peptone, 2% glucose.
YPG agar plates: 1% yeast extract, 2% peptone, 2.5% glycerol, 2% agar.
YPDA4% agar plates: 1% yeast extract, 2% peptone, 4% glucose, 2% agar.
BYTA media: 1% yeast extract, 2% Bacto tryptone, 1% potassium acetate, 50 mM potassium phthalate.
SPO media: 0.3% potassium acetate, pH 7.0.
YES media: 0.5% yeast extract, 3% glucose, 225 mg/L supplements.

Equipment and Reagents:
37% formaldehyde solution for molecular biology.
2.5 M glycine: Dissolve 93.8 g glycine in ddH2O (may require gentle heating) and bring up to 500 ml with ddH2O.
Diluent buffer: 0.143 M NaCl, 1.43 mM EDTA, 71.43 mM Hepes–KOH pH 7.5.
TBS buffer: 20 mM Tris–HCl pH 7.5, 150 mM NaCl.
2× FA lysis buffer: 100 mM Hepes–KOH pH 7.5, 300 mM NaCl, 2 mM EDTA, 2% Triton X-100, 0.2% Na-deoxycholate.
FastPrep screw-cap tubes.
100 mM PMSF.
Protease inhibitor tablets Complete EDTA free.
Zirconia/Silica beads 0.5 mm diameter.
FastPrep-24 5G Homogenizer.
Bioruptor Twin.
Dynabeads Protein G.
Magnetic rack.
ChIP Wash buffer 1—low salt: 1× FA lysis buffer, 0.1%SDS, 275 mM NaCl.
ChIP Wash buffer 2—high salt: 1× FA lysis buffer, 0.1%SDS, 500 mM NaCl.
ChIP Wash buffer 3: 10 mM Tris–HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40. 0.5% Na-deoxycholate.
ChIP Wash buffer 4 (TE): 10 mM Tris–HCl pH 8.0, 1 mM EDTA.
Chelex 100 Resin.
10 mg/ml Proteinase K
TES buffer: 50 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS.
Nuclease-free molecular biology grade water.
Filter tips.
Luna Universal Probe qPCR Master Mix.
LightCycler 480 Multiwell Plate 96.
LightCycler real-time PCR.
Qiagen purification kit.
LoBind DNA microcentrifuge tubes.
Quick blunting kit.
AMPure XP beads.
Klenow 3′ to 5′ exo minus.
Quick ligation kit (T4 DNA ligase).
NEXTflex DNA Barcodes—12 (Bioo Scientific; #NOVA-514102).
Phusion High-Fidelity DNA polymerase.
DynaMag-PCR magnet.
WizardSV Gel and PCR cleanup system.
Qubit dsDNA-HS Assay kit (Invitrogen).
Qubit Fluorometric Quantitation machine.
Agilent 2100 Bioanalyzer system.
High Sensitivity DNA Reagents kit (Agilent Technologies).
High Sensitivity DNA Chips (Agilent Technologies).
MiniSeq High throughput Reagent Kit (150-cycle) (Illumina).
Illumina Mini-seq.

Safety warnings

For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).

Formaldehyde and PMSF are toxic if inhaled, ingested or absorbed through the skin. Always wear a lab coat and gloves, and work in a chemical hood.

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