Sep 22, 2021

Public workspaceImmunoprecipitation, Decross-linking, and DNA Extraction

DOI
dx.doi.org/10.17504/protocols.io.bn3xmgpn
  • 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.bn3xmgpn
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. Immunoprecipitation, Decross-linking, and DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bn3xmgpn
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: 43863
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.
Immunoprecipitation, Decross-linking, and DNA Extraction
Immunoprecipitation, Decross-linking, and DNA Extraction
5m
5m
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in Amount1 mL ice-cold 1× FA buffer containing 0.1% SDS, Concentration1 millimolar (mM) PMSF and protease inhibitors with rotation for Duration00:05:00 .
Place the microcentrifuge tubes on a magnet and discard the supernatant. (1/3)

5m
Wash
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in Amount1 mL ice-cold 1× FA buffer containing 0.1% SDS, Concentration1 millimolar (mM) PMSF and protease inhibitors with rotation for Duration00:05:00 .
Place the microcentrifuge tubes on a magnet and discard the supernatant. (2/3)
5m
Wash
Prewash (n × 15) μl of Protein G Dynabeads (n = number of IP samples) in Amount1 mL ice-cold 1× FA buffer containing 0.1% SDS, Concentration1 millimolar (mM) PMSF and protease inhibitors with rotation for Duration00:05:00 .
Place the microcentrifuge tubes on a magnet and discard the supernatant. (3/3)
5m
Wash
Add the antibody against the protein of interest to Amount1 mL chromatin extract (step 13 in protocol "Cell Lysis and Sonication") and Amount15 µL prewashed Dynabeads and mix.

Note
The amount of SDS in immunoprecipitation can interfere with antibody binding efficiency therefore, lower amount of SDS can also be used. Either omit SDS in 2xFA buffer or reduce SDS added afterward to a final concentration of 0.05% before chromatin immunoprecipitation.

Note
The amount of antibody added should be in excess of the protein being immunoprecipitated and should be determined empirically. We use the following amounts of antibodies per IP:
Amount10 µL 9E10 (Tonbo Biosciences)
Amount7.5 µL 12CA5 (Roche)
Amount10 µL SV5-PK1 (Bio-Rad)
Amount10 µL anti-GFP (Roche)
Amount5 µL M2 FLAG (Sigma)

No blocking and preclearing is required for the magnetic beads.

Pipetting
Mix
Incubate on a rotating wheel at Temperature4 °C DurationOvernight .

5m
Incubation
Overnight
Place the tubes on the magnet and discard the supernatant. Perform the following washes, using 1 ml per sample of Wash buffer on a rotating wheel for 5 min at room temperature. Discard supernatants after each wash.
Wash
Wash using Amount1 mL ChIP Wash buffer 1 - low salt on a rotating wheel for Duration00:05:00 at TemperatureRoom temperature . Discard supernatant after wash.
5m
Wash
Wash using Amount1 mL ChIP Wash buffer 2 - high salt on a rotating wheel for Duration00:05:00 at TemperatureRoom temperature . Discard supernatant after wash.
5m
Wash
Wash using Amount1 mL ChIP Wash buffer 3 on a rotating wheel for Duration00:05:00 at TemperatureRoom temperature . Discard supernatant after wash.
5m
Wash
Wash using Amount1 mL ChIP Wash buffer 4 on a rotating wheel for Duration00:05:00 at TemperatureRoom temperature . Discard supernatant after wash.
5m
Wash
Following the final wash, place the samples on magnetic rack and discard the supernatant without disturbing the beads.
Pipetting
Move forward with:
  1. reversal of cross-linking and isolation of DNA for qPCR, OR
  2. ChIP-seq preparation after the final wash
Step case

Reverse cross-linking and isolate DNA for qPCR
12 steps

Use Chelex 100 as previously described [14].
Add Amount0.2 mL 10% slurry (wt/vol) in sterile water Chelex-100 resin directly to the washed Dynabeads (IP sample) and to Amount10 µL thawed “Input” samples (see step 14 from protocol "Cell Lysis and Sonication"). Keep the Chelex beads in suspension while pipetting.

Pipetting
Briefly vortex samples to mix the slurry and boil for Duration00:10:00 .

10m
Mix
Cool the tubes to TemperatureRoom temperature and quickly centrifuge condensate to the bottom of the tube at Centrifigation400 x g .
Proceed immediately to the next step.
Note
Variation in the amount of beads added can affect the specific signal–background ratio. Make sure to keep the slurry suspended while distributing.

Centrifigation
Add Amount2.5 µL Proteinase K (10 mg/ml) to each sample and incubate on a heat block at Temperature55 °C for Duration00:30:00 with occasional mixing to resuspend beads.

30m
Incubation
Mix
Boil the samples at Temperature100 °C for Duration00:10:00 to inactivate the Proteinase K.

10m
Spin the tubes briefly at Centrifigation400 x g and carefully transfer approximately Amount120 µL DNA supernatant , termed the IP sample in a new tube.
Note
Make sure not to transfer any Chelex resin as it can lead to loss of PCR signal.

Centrifigation
Store samples at Temperature-20 °C . (PAUSE POINT).

Pause
Purified DNA from step 12 can be used in qPCR, following the subsequent steps. We use Luna Universal Probe qPCR Master Mix in a 10 μl reaction in a 96-well plate and run the following program:
Set up the following reaction mix:

Amount3 µL DNA Template (for Input use 1:300, for IP use 1:6 dilutions)
Amount0.125 µL primer pair (20 μM each)
Amount5 µL master mix
ddH2O up to 10 μl final volume
Run the following program:

StepTemperatureTimeCycles
Initial denaturation95 °C1 min
Denaturation95 °C15 sec42 cycles
Extension60 °C30 sec
Melt curveaccording to Lightcycler 480 recommendations


Perform data analysis using Microsoft Excel software using the following formulas:

Determine the ChIP enrichment (i.e., ChIP/Input value), by calculating ΔCt using the following formula:
From this, the ChIP enrichment is calculated using:
where Ct values are mean threshold cycles of PCR performed in triplicate per DNA sample.

Note
To increase accuracy and reproducibility, we advise the use of an electronic dispensing pipette for qPCR. For primer design we recommend the use of Prime3Plus software. In the general settings of the software, we use product size range 70–200 bp (optimal amplicon size ~120 bp), optimal primer size 20 bp in length and optimal primer Tm for use with Luna Universal Probe qPCR Master Mix should be designed to anneal at 60 °C, with optimal primer GC% between 50% and 60%. Once primers are designed determine their efficiency and specificity using genomic DNA prior to performing qPCR with ChIP DNA sample. Finally, also include a No Template Control (NTC) in the reaction, where no amplification and no melting curve should be generated.

Computational step
To determine whether the amount of immunoprecipitated DNA is sufficient for library preparation for ChIP-seq, quantify Amount3 µL ChIP products and Input using the Qubit HS kit.
Note
Most commonly, immunoprecipitated DNA yields are in the range of 2–10 ng.