Sep 22, 2021

Public workspaceGrowth Conditions for SMC Proteins

DOI
dx.doi.org/10.17504/protocols.io.bn3imgke
  • Adele L. Marston1,
  • Daniel Robertson1,
  • Vasso Makrantoni1
  • 1The Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences University of Edinburgh Edinburgh UK
  • Springer Nature Books
Satyavati Kharde
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DOI: dx.doi.org/10.17504/protocols.io.bn3imgke
External link: https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#Abs1
Protocol Citation: Adele L. Marston, Daniel Robertson, Vasso Makrantoni 2021. Growth Conditions for SMC Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.bn3imgke
License: This is an open access protocol 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
Created: October 27, 2020
Last Modified: September 22, 2021
Protocol Integer ID: 43850
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.

Chromatin immunoprecipitation (ChIP) is a powerful method for assaying protein–DNA binding in vivo and is broadly used to estimate the density of DNA-bound proteins at specific sites in the genome. ChIP is a multistep assay and every step needs to be optimized for consistent results. Briefly, protein–DNA associations are immobilized by cross-linking with formaldehyde [1,2,3] before shearing the chromatin, either mechanically [4] or by enzymatic digestion [5] into DNA fragments of average size 200–500 bp. Specific cross-linked protein–DNA complexes are then isolated by immunoprecipitation using an antibody to the protein of interest. Finally, the cross-links are reversed, and the retrieved DNA is analyzed to determine the sequences bound by the protein. ChIP followed by quantitative real-time PCR (ChIP-qPCR), using specific primers, can be used to measure protein association and relative abundance at a particular genomic locus. Alternatively, ChIP can be combined with next generation sequencing (ChIP-seq) to provide a genome-wide view of protein occupancy. While ChIP-seq allows for relative protein abundance at distinct chromosomal addresses to be compared within a sample, differences between samples cannot be quantified without introducing a method to normalize. Typically, this involves “spike in” of a known amount of DNA or cross-linked cells from a different species, with sufficient sequence divergence from the organism of interest to allow sequencing reads to be confidently distinguished bioinformatically [6,7,8]. This technique, referred to as calibrated ChIP-seq, makes it possible to quantitate genome-wide changes in the distribution of an epitope tagged protein and allows for quantification of differences in occupancy between experimental samples [8]. Calibrated ChIP-seq requires that both calibration and experimental organisms carry the same epitope tag and can be immunoprecipitated by the same protocol. For this protocol we use S. pombe to calibrate S. cerevisiae, a combination that also allows us to invert the roles, that is, calibrate S. pombe with S. cerevisiae.
The ChIP method described here has been optimized for use with chromatin from two species of yeast,S. cerevisiae and S. pombe; however, it should be easy to adapt it for use with other chromatin sources. To demonstrate the robustness of our ChIP and library preparation protocols we performed ChIP against the Scc1 subunit of the cohesin multiprotein complex, tagged with the 6HA epitope [9,10,11] . We have also used a similar protocol for the condensin subunit Brn1 [12] and for the meiotic counterpart of cohesin, Rec8 [13]. Here, we outline in detail an optimized protocol for cross-linking and harvesting cells, fragmenting chromatin, immunoprecipitating the desired protein–DNA complexes, and preparing the library for sequencing on the Illumina MiniSeq platform. A schematic stepwise representation of the method is illustrated in Fig.1.



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Acknowledgements:
We are grateful to Manu Shukla for discussions and comments on the ChIP-seq library preparation and for kindly providing a representative Bioanalyzer image, Nicholas Toda, Jesus Torres-Garcia, and Flora Paldi for sharing tips on the ChIP-seq library protocol and Stefan Galangher and Lesley Clayton for general comments on the manuscript. This work was funded by Wellcome through a Senior Research Fellowship to AM and a Wellcome Centre core grant [107827 and 203149].

Guidelines
Chromatin immunoprecipitation (ChIP) is broadly used to study chromatin dynamics. Changes in occupancy of chromosomal proteins at specific loci within the genome can be measured by using ChIP-qPCR. However, this technique is costly and time consuming with high variability per experiment. Alternatively, ChIP-seq can be used to measure differences in a protein’s occupancy genome wide. Finally, calibrated ChIP-seq is essential when measuring changes in occupancy between different experimental samples.

Here we describe an optimized ChIP protocol for yeast SMC proteins that can be completed within 3 days for samples analyzed by qPCR and 4 days for samples to be further processed by calibrated deep sequencing. The protocol encompasses five distinct steps: cross-linking and cell harvesting; cell lysis and sonication; immunoprecipitation, decross-linking and DNA extraction and finally determination of the size and DNA concentration of sonicated samples. These five steps are outlined here.
Materials
Yeast Strains and Growth Material:
  1. 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].
  2. 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).
  3. 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).
  4. YPDA media: 1% yeast extract, 2% peptone, 2% glucose.
  5. YPG agar plates: 1% yeast extract, 2% peptone, 2.5% glycerol, 2% agar.
  6. YPDA4% agar plates: 1% yeast extract, 2% peptone, 4% glucose, 2% agar.
  7. BYTA media: 1% yeast extract, 2% Bacto tryptone, 1% potassium acetate, 50 mM potassium phthalate.
  8. SPO media: 0.3% potassium acetate, pH 7.0.
  9. YES media: 0.5% yeast extract, 3% glucose, 225 mg/L supplements.

Equipment and Reagents:
  1. 37% formaldehyde solution for molecular biology.
  2. 2.5 M glycine: Dissolve 93.8 g glycine in ddH2O (may require gentle heating) and bring up to 500 ml with ddH2O.
  3. Diluent buffer: 0.143 M NaCl, 1.43 mM EDTA, 71.43 mM Hepes–KOH pH 7.5.
  4. TBS buffer: 20 mM Tris–HCl pH 7.5, 150 mM NaCl.
  5. 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.
  6. FastPrep screw-cap tubes.
  7. 100 mM PMSF.
  8. Protease inhibitor tablets Complete EDTA free.
  9. Zirconia/Silica beads 0.5 mm diameter.
  10. FastPrep-24 5G Homogenizer.
  11. Bioruptor Twin.
  12. Dynabeads Protein G.
  13. Magnetic rack.
  14. ChIP Wash buffer 1—low salt: 1× FA lysis buffer, 0.1%SDS, 275 mM NaCl.
  15. ChIP Wash buffer 2—high salt: 1× FA lysis buffer, 0.1%SDS, 500 mM NaCl.
  16. 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.
  17. ChIP Wash buffer 4 (TE): 10 mM Tris–HCl pH 8.0, 1 mM EDTA.
  18. Chelex 100 Resin.
  19. 10 mg/ml Proteinase K
  20. TES buffer: 50 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS.
  21. Nuclease-free molecular biology grade water.
  22. Filter tips.
  23. Luna Universal Probe qPCR Master Mix.
  24. LightCycler 480 Multiwell Plate 96.
  25. LightCycler real-time PCR.
  26. Qiagen purification kit.
  27. LoBind DNA microcentrifuge tubes.
  28. Quick blunting kit.
  29. AMPure XP beads.
  30. Klenow 3′ to 5′ exo minus.
  31. Quick ligation kit (T4 DNA ligase).
  32. NEXTflex DNA Barcodes—12 (Bioo Scientific; #NOVA-514102).
  33. Phusion High-Fidelity DNA polymerase.
  34. DynaMag-PCR magnet.
  35. WizardSV Gel and PCR cleanup system.
  36. Qubit dsDNA-HS Assay kit (Invitrogen).
  37. Qubit Fluorometric Quantitation machine.
  38. Agilent 2100 Bioanalyzer system.
  39. High Sensitivity DNA Reagents kit (Agilent Technologies).
  40. High Sensitivity DNA Chips (Agilent Technologies).
  41. MiniSeq High throughput Reagent Kit (150-cycle) (Illumina).
  42. 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.
Growth Conditions for SMC Proteins
Growth Conditions for SMC Proteins
3d 18h 45m
3d 18h 45m

Note
S. cerevisiae strains for mitotic studies are grown in YPDA at Temperature25 °C . The most consistent results, at least for cohesin, are obtained when cells are arrested in metaphase of mitosis prior to the ChIP procedure. This can be achieved either by depletion of the anaphase-promoting complex subunit, Cdc20, or treatment of the cells with the microtubule-depolymerizing drug nocodazole.

For depletion of Cdc20, use a construct where CDC20 is under control of the methionine-repressible promoter, pMET3 (pMET3-CDC20).
Briefly for Cdc20 depletion, dilute an overnight culture to OD600 = 0.2 in minimal media lacking methionine and grow for Duration01:00:00 –Duration02:00:00 at Temperature25 °C to OD600 = 0.3–0.4.
3h
Incubation
Dilute culture back to OD600 = 0.2 in same media and arrest cells in G1 by adding Concentration5 microgram per milliliter (μg/mL) α-factor for Duration01:30:00 and Concentration2.5 microgram per milliliter (μg/mL) α-factor for an additional Duration01:30:00 .

3h
Incubation
Check microscopically that at least 90% of cells are arrested before collecting on a filter (Whatman ME25, 0.45 μm), and wash with 10 volumes of medium lacking sugar with the aid of a vacuum pump.
Wash
Quickly resuspend cells in YPDA containing Concentration8.5 millimolar (mM) methionine and re-add methionine to Concentration4 millimolar (mM) every Duration00:45:00 .
45m
Harvest cells after Duration02:00:00 –Duration02:30:00 in a metaphase arrest confirmed by microscopy.
4h 30m
For nocodazole arrest, follow these subsequent steps:
Dilute an overnight culture to OD600 = 0.2 in YPDA and grow for Duration01:00:00 –Duration02:00:00 at Temperature25 °C to OD600 = 0.3–0.4.
3h
Incubation
Dilute culture back to OD600 = 0.2 in YPDA media containing a mixture of Concentration15 microgram per milliliter (μg/mL) nocodazole and Concentration30 microgram per milliliter (μg/mL) benomyl .

Read nocodazole every Duration01:00:00 at Concentration7.5 microgram per milliliter (μg/mL) . Harvest cells after Duration02:00:00 –Duration02:30:00 confirming metaphase arrest by microscopy.

5h 30m
For inducing meiosis, follow these subsequent steps:
Note
For studies of protein occupancy during meiosis we use diploid S. cerevisiae strains of SK1 background including (a)REC8-3HA ndt80Δ (AM4015), as previously described [13] and (b)REC8-6HIS-3FLAG (AM11000).

Recover SK1 strains from Temperature-80 °C on YPG agar plates DurationOvernight at Temperature30 °C , before transferring to YPDA4% agar plates for a further Duration12:00:00 -Duration30:00:00 at Temperature30 °C .
1d 18h
Overnight
Inoculate cultures in liquid YPDA at Temperature30 °C with shaking for ~Duration24:00:00 , prior to inoculating into BYTA medium to OD600 = 0.3 DurationOvernight .
1d
Overnight
The next morning, spin cells down, wash with dH2O and resuspend in SPO medium to OD600 = 1.8 and shake at Temperature30 °C .
Centrifigation
Wash
For prophase I arrest (ndt80Δ) for Rec8 cells, harvest Amount50 mL media Duration06:00:00 after resuspension in sporulation medium and confirm arrest by FACS.
Note
S. pombe strains used for calibration are listed in the materials under "Yeast Strains and Growth Material" and are grown in YES at Temperature30 °C .

6h
Incubation