Chromatin Immunoprecipitation (ChIP)

Original protocol published in Malyavko, A.N., Petrova, O.A., Zvereva, M.I. et al. Functional duplication of Rap1 in methylotrophic yeasts. Sci Rep 9, 7196 (2019). https://doi.org/10.1038/s41598-019-43595-8

With modifications described in Alexander N Malyavko, Olga A Petrova, Maria I Zvereva, Vladimir I Polshakov, Olga A Dontsova (2022) Telomere length regulation by Rif1 protein from Hansenula polymorpha eLife 11:e75010. https://doi.org/10.7554/eLife.75010

This protocol was adapted from the ChIP protocol described by the de Lange laboratory (https://delangelab.org/protocols)

Grow yeast cells in 100 ml of YPD at 37 °C to OD₆₀₀ ~0.9.
Add formaldehyde to a final concentration of 1%, incubate for 30 min at 25 °C. (Cell fixation)
Add glycine to a final concentration of 125 mM final, incubate 5 min at 25 °C. (To stop crosslinking).
Harvest cells by centrifugation (3000 g, 5min), wash 3x with TBS.
Re-suspend cells in 0.5 ml of buffer A, transfer to VK-05 tubes (Bertin Instruments).
Lyse cells with glass beads in a Precellys Evolution homogenizer (Bertin Instruments): 8 cycles of (20s 7200 rpm then 1 min on ice).
Add 0.5 ml of buffer B. Sonicate the lysates to shear chromatin (~300-500 bp median fragment size).
Clear the lysate by centrifugation (14000 g, 15 min).
Save a 7.5 mkl aliquot of the lysate (this will become the ‘1% of input’ sample).
Add 20 µl of Pierce anti-HA magnetic beads (Thermo Scientific) to 0.75 ml of the lysates and incubate (gentle rotation) at +4 °C for 2 hours.
Wash the beads:
1. 1x with 1 ml of Buffer C (3-5 min of gentle rotation at room temperature).
2. 1x with 1 ml of Buffer D (3-5 min of gentle rotation at room temperature).
3. 2x with 1 ml of Buffer E (3-5 min of gentle rotation at room temperature).
4. 1x with 1 ml of TE buffer (3-5 min of gentle rotation at room temperature).
Elution:
1. add 250 μl of 0.1 M NaHCO₃, 1% SDS and incubate at 65 °C for 15 min (vigorous shaking). Collect the eluate.
2. add 250 μl of 0.1 M NaHCO₃, 1% SDS and incubate at room temperature for 10 min (occasional shaking). Collect the eluate.
Combine the two eluate fractions (the ‘IP’ sample), add NaCl (to a final concentration of 0.2 M) and incubate overnight at 65 °C (to reverse cross-linking).
Add 500 mkl of 0.1 M NaHCO₃, 1% SDS to the ‘1% of input’ sample, add NaCl (to a final concentration of 0.2 M) and incubate overnight at 65 °C (to reverse cross-linking).
In the morning, add 20 μl of 1 M Tris pH 6.5, 10 μl of 0.5 M EDTA and 30 μg of RNase A and incubate at 37 °C for 30 min.
Add 100 μg of proteinase K and incubate at 37 °C for 1 hour.
Extract the DNA with phenol/chloroform, precipitate with EtOH and dissolve in 25 μl of TE buffer.
Real-time PCR was performed in a 15 μl mixture containing 0.45 μl of DNA (either ‘IP’ or ‘1% of input’ sample), 1x Taq KCl buffer, 2.5 mM MgCl₂, 0.6 μM primers, 0.2 mM dNTPs, 0.4x SYBR Green and 0.9 U of Taq polymerase (Thermo Fisher Scientific).
Primers:
1. TELf GCTAGGGAGGTGTGGGTAGTGG
2. TELr CGCCTGCCCCTACATTACTTTC
3. ALA1f GGGTTCCACCACAGAACTCAAC
4. ALA1r TTCGGAGAGACCTACCCAGACC

Buffers:

TBS: 50 mM Tris, pH 7.6; 150 mM NaCl

Buffer A: 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.01% sodium deoxycholate and 1x Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific)

Buffer B: 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1.9% Triton X-100, 0.19% sodium deoxycholate and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific)

Buffer C: 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate

Buffer D: 50 mM HEPES pH 7.5, 0.5 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate

Buffer E: 10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate

TE buffer: 10 mM Tris pH 8, 1 mM EDTA