Sep 22, 2021

Public workspaceDetermine the Size of Sonicated Samples and the DNA Concentration

DOI
dx.doi.org/10.17504/protocols.io.bn33mgqn
  • 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
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bn33mgqn
External link: https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10
Protocol Citation: Vasso Makrantoni, Daniel Robertson, Adele L. Marston 2021. Determine the Size of Sonicated Samples and the DNA Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.bn33mgqn
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 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: 43867
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.
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.
Before start
Use sonicated chromatin samples (step 15 from "Cell Lysis and Sonication") to determine the fragment size.
Determine the Size of Sonicated Samples and the DNA Concentration
Determine the Size of Sonicated Samples and the DNA Concentration
3h 0m 30s
3h 0m 30s
To a Amount100 µL Input sample add Amount80 µL TE buffer containing Concentration300 millimolar (mM) NaCl .

Pipetting
Decross-link at Temperature65 °C DurationOvernight .

30s
Overnight
Add Amount2 µL RNase A (10 mg/ml) and incubate at Temperature37 °C for Duration01:00:00 .
Note
RNase treatment is important as high levels of RNA will interfere with DNA purification when using commercially available PCR purification kits. DNA yield can be markedly reduced as the columns become saturated.


1h
Incubation
Pipetting
Add Amount20 µL Proteinase K (10 mg/ml) and incubate at Temperature65 °C for Duration02:00:00 .

2h
Incubation
Pipetting
Purify DNA using a PCR purification kit. Run purified DNA on a 2% agarose gel with a 100 bp DNA ladder marker to determine fragment size. Ideally sonication should yield an enrichment of fragments between 200 and 400 bp (Fig. 2a).
Fig. 2
(a) Representative image of mitotic yeast cells sonicated with a Bioruptor Twin (Diagenode) for a 30-min round (power setting: High, 30 s ON/30 s OFF). DNA from two different samples was loaded on a 2% agarose gel with a 100 bp marker ladder.
(b) Representative optimal BioAnalyzer trace (upper panel) and contaminated trace with adapter (bottom panel)
(c). Examples of profiles generated by chromatin immunoprecipitation followed by sequencing (ChIP–seq) of the cohesin subunit Scc1 in wild-type cells (IP shown in blue; Input shown in grey) and calibrated withS. pombeScc1 distribution in representative chromosome V (IP shown in blue, bottom panel)

PCR
Measure DNA concentration using a Qubit HS assay kit.
Computational step