A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae

Materials and Reagents

1. 50 ml centrifuge FALCON-type tubes
2. 1.5 ml microtubes
3. Cuvettes
4. Glass beads (Retsch, catalog number: 22.222.0004)
5. anti-Histone H3 (Abcam, catalog number: ab1791)
6. anti-Pgk1 (Invitrogen, catalog number: 459250)
7. Anti-Rabbit IgG (Sigma-Aldrich, catalog number: A0545)
8. Ani-Mouse IgG (Bio-Rad, catalog number: 170-6516)
9. Cell culture media:
   Peptone (FORMEDIUM, catalog number: PEPCFG)
   Glucose (FORMEDIUM, catalog number: GLUCFG)
   Galactose (FORMEDIUM, catalog number: GALCFG)
   Yeast extract (FORMEDIUM, catalog number: YEACFG)
   Yeast nitrogen base (CONDA, catalog number: 1553)
   Ammonium sulfate (PanReac AppliChem, catalog number: 141140)
   Uracil (Sigma-Aldrich, catalog number: U-1128)
   L-Methionine (FORMEDIUM, catalog number: DOC0168)
   L-Tryptophan ((FORMEDIUM, catalog number: DOC0187)
   Adenine sulphate (FORMEDIUM, catalog number: DOC0229)
   L-Leucine (SIGMA ALDRICH, catalog number: L8912)
   L-Lysine monohydrate (FORMEDIUM, catalog number: DOC0159)
   L-Histidine (FORMEDIUM, catalog number: DOC0144)
10. MilliQ H₂O
11. HEPES (iNtRON BIOTECHNOLOGY, catalog number: 1017365)
12. KCl (PanReac AppliChem, catalog number: 131494)
13. NaCl (PanReac AppliChem catalog number: 121659)
14. MgCl₂ (Sigma-Aldrich, catalog number: M9272)
15. CaCl₂ (PanReac AppliChem catalog number: 131232)
16. Triton X-100 (PanReac AppliChem, catalog number: 142314)
17. Sucrose (Sigma-Aldrich, catalog number: S9378)
18. Spermidine (Sigma-Aldrich, catalog number: S2626)
19. Spermine (Sigma-Aldrich, catalog number: S3256)
20. EDTA (PanReac AppliChem, catalog number: 131669)
21. Glycerol (PanReac AppliChem, catalog number: 141339)
22. IGEPAL CA-630 (NP-40) (Sigma-Aldrich, catalog number: I8896)
23. Urea (Sigma-Aldrich, catalog number: U-6504)
24. DTT (FORMEDIUM, catalog number: DTTCFG)
25. PMSF (Sigma-Aldrich, catalog number: P7626)
26. Tris (PanReac AppliChem, catalog number: 141940)
27. SDS (BioPure, catalog number: 8030S)
28. Bromophenol blue (Sigma-Aldrich, catalog number: B5525)
29. Β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148)
30. N-butyric acid (Sigma-Aldrich, catalog number: B103500)
31. Buffer 1 and Buffer 2 (see Recipes)
32. Sample Buffer (see Recipes)
33. Cell culture media (see Recipes)
    1. Yeast complete medium YPD
    2. Synthetic dropout medium (SD)

Equipment

1. 500 ml Erlenmeyer glass flasks
2. Refrigerated-regulated centrifuges (for 50 ml tubes: Hettich, model: Universal 32R; for 1.5 ml tubes: Beckman Coulter, model: Allegra 21R)
3. Spectrophotometer: Ultrospec 10 Cell Density Meter (Amersham Biosciences)
4. Vortex (Genie 2 with TurboMix Attachment, a multisample device)
5. Orbital shaker/ incubator for yeast culture: Multitron Standard (Infors AG)

Procedure

1. Saccharomyces cerevisiae standard culture conditions
   1. Saccharomyces cerevisiae can be grown in yeast complete medium (YPD) (Recipe 3a) or in Synthetic dropout medium (SD) (Recipe 3b) supplemented with strain-specific requirements.
   2. Inoculate 150 ml of culture medium (containers of 500 ml) with S. cerevisiae cells from a 5 ml preculture growing exponentially, and incubate them at 30 °C (or at the required temperature). Cultures must be shaken at 180-200 rpm on an orbital shaker.
   3. Let cultures grow until an OD₆₀₀ ~0.7-0.8 is reached.
   4. Transfer cultures to 50 ml centrifuge tubes (x3) and harvest them by centrifugation (2,500 x g for 2 min at 4 °C). Discard the supernatant.
   5. In this step, pellets can be stored at -80 °C or can be used to continue the procedure.
      
      
2. Chromatin purification protocol
   1. All the subsequent steps are performed at 4 °C.
   2. Wash cells with 5 ml of sterile MilliQ H₂O, centrifuge (2,500 x g for 2 min) and resuspend the pellet in 1 ml of MilliQ H₂O.
   3. Transfer to 1.5 ml microtubes and centrifuge them at 2,500 x g for 1 min at 4 °C. Discard the supernatant.
   4. Resuspend the pellet in 200 µl of Buffer 1 (Recipe 1 Table 1) and add 200 µl of glass beads (size: 425-600 µm).
   5. Lyse cells by continuous vigorous vortexing for 4 min at maximal speed using a Genie 2 vortex with a TurboMix Attachment (a multisample device) at 4 °C.
   6. Centrifuge at 500 x g for 5 min to spin down the glass beads and intact cells. Transfer the supernatant to a new 1.5 ml microtube. Be careful to not take anything from the pellet to efficiently reduce cytoplasmic contamination during subsequent steps.
   7. Centrifuge the supernatant at 500 x g for 5 min to discard the residual intact cells. Transfer the supernatant to a new 1.5 ml microtube. The pellet’s contamination in this step leads to cytoplasmic contaminants in the final purified chromatin fraction.
   8. Keep 5-10 µl of supernatant as the input control.
   9. Centrifuge the rest of the supernatant at high speed (20,000 x g) for 20 min.
   10. Discard the supernatant using a pipette and resuspend the visible pellet (P1) in 200 µl of Buffer 1 (Table 1).
   11. Centrifuge at high speed (20,000 x g) for 20 min.
   12. Discard the supernatant and resuspend the visible pellet (P2) in 200 µl of Buffer 2 (Recipe 1 Table 2).
   13. Centrifuge at high speed (20,000 x g) for 20 min.
   14. Discard the supernatant. The visible final pellet (P3) constitutes the chromatin-enriched fraction.
   15. For SDS-PAGE and Western blots, resuspend P3 in 20 µl of 1x Tris-Glycine SDS Sample Buffer (Recipe 2) and incubate it for 10 min at 100 °C. After boiling, spin down at 30,000 x g for 1 min. Use the supernatant for gel electrophoresis.
   16. For the mass spectrometry analysis, gently resuspend the chromatin-enriched fractions (P3) in 20 µl of Tris-HCl 50 mM, pH 7.5, at room temperature. It can be frozen at -20 or -80 °C for further analyses.
       
       
       
3. Scale-up conditions
   1. To scale up the protocol, 600 ml of S. cerevisiae cultures at OD₆₀₀ ~0.7-0.8, must be used and the complete protocol must be performed at 4 °C.
   2. Harvest the culture into three centrifuge tubes and centrifuge at 2,500 x g for 2 min. Under these conditions, each tube contains a cell pellet corresponding to 200 ml of the culture cells.
   3. Wash each cell’s pellet with 5 ml of sterile MilliQ H₂O, centrifuge at 2,500 x g for 2 min and resuspend each pellet in 1 ml of MilliQ H₂O.
   4. Transfer to 1.5 ml microtubes and centrifuge at 2,500 x g for 1 min. Discard the supernatant.
   5. Resuspend each pellet in 200 µl of Buffer 1 (Table 1) and add 200 µl of glass beads (425-600 µm).
   6. Lyse cells by vigorous continuous vortexing for 4 min (Genie 2 with the TurboMix Attachment, a multisample device, at maximal speed).
   7. Centrifuge at 500 x g for 5 min to spin down the glass beads and intact cells. Transfer the three supernatants corresponding to the same cell culture to a new single 1.5 ml microtube.
   8. Subsequent steps are similar to the standard protocol. In Steps B10 and B12 of the standard protocol, scale up buffers to 600 µl.
   9. For SDS-PAGE and Western blots, resuspend P3 in 60 µl of 1x Tris-Glycine SDS Sample Buffer (Recipe 2; Table 3) and incubate the desired amount for 10 min at 100 °C. After boiling, spin down at 30,000 x g for 1 min. Use the supernatant for gel electrophoresis.
   10. For the mass spectrometry analysis, gently resuspend the chromatin-enriched fractions (P3) in 20 µl (or that required) of Tris-HCl 50 mM, pH 7.5, at room temperature. It can be frozen at -20 or -80 °C for further analyses.
       
       
       
4. Scale-down conditions
   1. To scale down the protocol, 50 ml of S. cerevisiae cultures at OD₆₀₀ ~0.7-0.8, are used and all the protocol must be performed at 4 °C.
   2. When scaling down the protocol, the solution volumes used for the standard procedures (200 μl) are maintained to easily manipulate samples.
   3. For SDS-PAGE and Western blots, resuspend P3 in 20 µl of 1x Tris-Glycine SDS Sample Buffer (Recipe 2; Table 3) and incubate the desired amount for 10 min at 100 °C. After boiling, spin down at 30,000 x g for 1 min. Use the supernatant for gel electrophoresis.

Data analysis

The protein analysis of the different fractions from the yChEFs procedure is shown in Figure 1.


Figure 1. Protein analysis of the different fractions from the yChEFs procedure. Histone H3 and Pgk1, as controls, were analyzed by Western blot (Anti-Rabbit IgG as a secondary antibody for anti-H3 primary antibody and Anti-mouse IgG as a secondary antibody for anti-Pgk1 primary antibody). Note that chromatin was properly isolated as no cytoplasmic Pgk1 protein was detected. WCE: whole-cell (lysate). P1-P3: Pellet 1-3. S1-S3: Supernatant 1-3. The 1/10 volumes from each fraction were loaded, except for the whole P3 fraction.

Recipes

1. Buffer 1 and Buffer 2 (Tables 1 and 2)
   
   Table 1. Buffer 1 composition and preparation
   
   *FC: Final concentration
   
   Table 2. Buffer 2 composition and preparation
   
   *FC: Final concentration
   
   
2. Sample buffer (Table 3)
   
   Table 3. Sample buffer composition and preparation
   
   *FC: Final concentration
   
   
3. Cell culture media
   1. Yeast complete medium (YPD)
      2% (w/v) peptone
      2% (w/v) glucose (or galactose)
      1% (w/v) yeast extract
   2. Synthetic dropout medium (SD)
      0.17% (w/v) yeast nitrogen base
      0.5% (w/v) ammonium sulfate
      2% (w/v) glucose (or galactose)
      Supplemented requirements:
      L-Histidine 20 mg/L
      L-Lysine monohydrate 30 mg/L
      L-Leucine 150 mg/L
      L-Methionine 20 mg/L
      L-Tryptophan 20 mg/L
      Adenine sulphate 50 mg/L
      Uracil 20 mg/L

Acknowledgments

This protocol is described in our previous report (Cuevas-Bermúdez et al., 2019). This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness, MINECO and FEDER funds (BFU2016-77728-C3-2-P to F.N.) and the Junta de Andalucía (BIO258 to F. N.). A.C-B. is a recipient of an FPI predoctoral contract from MINECO. A.I.G-G was a recipient of MEC and a postdoctoral fellowship from the University of Jaén. F.G-S is a recipient of a predoctoral fellowship from the University of Jaén.

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