Chromatin Immunoprecipitation from Caenorhabditis elegans Somatic Cells

Abstract

Chromatin Immunoprecipitation is a regularly used method to detect DNA–protein interaction in diverse biological samples. Here we describe the application of ChIP for histone modifications in adult-stage Caenorhabditis elegans somatic cells.

1. Introduction

Chromatin Immunoprecipitation (ChIP) is a widely used method to detect the localization of protein factors on chromatin. The interactions between proteins and protein-DNA can be first stabilized by reversible cross-linking by formaldehyde [1] or UV light [2] treatment. Chromatin is then fragmented by sonication or enzyme digestion [3] to generate chromatin fragments. Chromatin fragments that associate with the specific protein mark are then captured by specific antibodyies. The DNA embedded in the chromatin fragment is then recovered by immunoprecipitation. Here we describe the application of ChIP for histone modifications in adult C. elegans somatic cells based on the ChIP method in [4].

2. Materials

2.1. Caenorhabditis elegans Collection

M9 buffer: 3.0 g KH₂PO₄, 11.32 g Na₂HPO₄•7H₂O, 5 g NaCl. Bring to 1 L with distilled water.

2.2. Chromatin Immunoprecipitation

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ cm at 25 °C). Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing of waste materials. We do not add sodium azide to reagents.

1. Phosphate-buffered saline (PBS; 10×): 100 mM Na₂HPO₄, 18 mM KH₂PO₄, 1.37 M NaCl, 27 mM KCl (see Note 1).

2. Protease inhibitors.

3. 1% formaldehyde in PBS: dilute 16% formaldehyde (methanol-free) with PBS to 1% (see Note 2).

4. 2 M glycine.

5. RNase A.

6. 10% sarkosyl.

7. Protein A-Agarose beads.

8. Elution buffer: 1% SDS in TE with 250 mM NaCl (see Note 3).

9. 10 mg/mL Proteinase K: dissolve proteinase K with water. Store the aliquots at −20 °C.

10. FA buffer: 50 mM HEPES–KOH (pH 7.5), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl (see Note 4).

11. FA-1 M salt buffer: 50 mM HEPES–KOH (pH 7.5), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 M NaCl.

12. FA-500 mM salt buffer: 50 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 500 mM NaCl.

13. TEL buffer: 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0.

3. Methods

3.1. C. elegans Collection

Wash germline-less adult worms off plates with cold M9. Transfer worms in M9 into 15 mL or 50 mL conical tube by using glass pipette. Put the conical tube on ice and wait ~10 min for worms to settle to the bottom of the tube. Aspirate off the M9. Repeat the washing step one more time with adequate amount of M9 to remove as much OP50 as possible. Resuspend worms with cold M9 and centrifuge at 16,400 × g for 30 s in a clinical centrifuge to pellet worms. Remove as much M9 as possible. The collection procedure should be finished within 30 min. Snap-freeze worms with liquid nitrogen and store the worm pellet at −80°C (seeNote 5).

3.2. Chromatin Immunoprecipitation

1. Thaw worms on ice. Resuspend worms with cold M9 and drop worm suspension into liquid nitrogen to make frozen worm beads (see Note 5).

2. Grind frozen worm beads into fine powder in liquid nitrogen with mortar and pestle. Inspect the fine powder with microscope. When worms are ground into “worm fragment,” stop grinding and transfer the fine powder into prechilled 50 mL conical tubes (see Note 6).

3. Thaw the fine powder on ice. Measure the sample volume (V1).

4. Add 10 × V1 of room temperature 1% formaldehyde in PBS into worm sample and incubate at room temperature for 10 min with gentle rocking.

5. Quench formaldehyde by adding glycine to a final concentration of 125 mM and incubating with gentle rocking for 5 min at room temperature.

6. Spin the sample at 4000 × g for 3 min at 4 °C. Wash the pellet with PBS (protease inhibitors added). Repeat this step two more times.

7. Resuspend pellet in FA buffer with protease inhibitors (see Note 7).

8. Sonicate the sample with Bioruptor (30 s ON and 1 min OFF) for 25–30 cycles (see Note 7).

9. Spin at 13,200 rpm (around 16,400 × g) for 15 min at 4 °C.

10. Quantitate total protein (see Note 8).

11. Incubate appropriate amount of worm lysate with antibody and 1% sarkosyl overnight at 4 °C with rotation (see Note 8).

12. Equilibrate Protein A-Agarose beads in FA buffer, preclear them with 10 mg/mL of BSA.

13. Set aside 10% of the worm lysate used in the IP in step 11, and store it at 4 °C to be used as “Input” the next day.

14. Next day, add 40 μL of precleared bead slurry (around 20 μL beads) to each IP, and incubate for 3 h at 4 °C with rotation.

15. Spin sample for 1–2 min at 16,400 × g at 4 °C.

16. Remove supernatant. Wash the beads with 1 mL of the following buffers with rotation:

    Twice with FA buffer + proteinase inhibitors for 5 min each at 4 °C.

    Once with FA-500 mM NaCl for 10 min at room temperature.

    Once with FA-1 M NaCl for 5 min at room temperature.

    Once with TEL buffer for 10 min at room temperature.

    Twice with TE for 5 min at room temperature.

17. Elute precipitated chromatin from protein A beads by adding 150 μL of elution buffer and incubate at 65 °C for 15 min. Every 5 min resuspend beads by flicking tube or quick vortex. Spin to collect the supernatant. Repeat elution step once. Combine the supernatants.

18. Add 300 μL of elution buffer to input samples from step 13.

19. Incubate ChIP samples from step 17 and Input samples from step 18 with 20 μg RNase A at 37 °C for 30 min.

20. Add 2 μL of 10 mg/mL proteinase K and incubate at 55 °C for1 h.

21. Reverse cross-link at 65 °C overnight (see Note 9).

22. Purify DNA by Qiagen PCR purification columns (QIAquick PCR Purification Kit) and elute with 30 μL elution buffer.

23. Proceed either to Q-PCR (see Note 10) or to amplification for library construction.

4. Notes

1. PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the reagents listed above in 800 mL of H₂O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H₂O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm²) on liquid cycle or by filter sterilization. Store PBS at room temperature.

2. Prepare the formaldehyde solution in fume hood. Follow the instruction for using formaldehyde in all applications requiring “freshly prepared” formaldehyde solution.

3. TE: 10 mM Tris–HCl (pH 8.0), 1 mM EDTA.

4. Prepare 500 mM EDTA stock. Weigh out 186.12 g of EDTA. Na₂•2H₂O (molecular weight: 372.24). Transfer to 2 L beaker/conical flask. Add 800 mL deionized/Milli-Q water. While stirring vigorously on a magnetic stirrer, add NaOH pellet or 10 N NaOH to adjust the solution pH 8.0. ~20 g NaOH pellet is required to adjust the pH 8.0. It is not easy to dissolve EDTA. To dissolve the EDTA completely, solution pH 8.0 is required. Adjust the volume to 1000 mL with deionized water. Mix it again. Transfer the solution to auto-clavable bottle. Sterilize the solution by autoclaving (20 min at 15 lb./sq.in. (psi) from 121–124 °C on liquid cycle).

5. For ChIP with protein factors instead of histones, the freezing step needs to be carried out right after cross-linking to preserve protein–protein and protein–DNA interactions.

6. Worms that are overground will be lost in the supernatant in step 6.

7. The volume of FA buffer and sonication cycle number are determined by sonication efficiency of the equipment. To determine the appropriate sonication condition, the sonicated chromatin needs to be processed through steps 18–21, and the size of the extracted DNA needs to be checked by electrophoresis. For histone modification ChIP, we use the sonication condition that produces DNA fragments ranging from 200 to 500 bp. We start with 10⁴ adult worms in 400 μL FA buffer.

8. The optimal antibody amount needs to be empirically determined by comparing the IP specificity and recovery efficiency using Q-PCR. Primers for specific and nonspecific regions are needed for the Q-PCR. The optimal antibody amount should yield the highest recovery at specific regions without causing increased recovery at nonspecific regions.

9. To avoid condensation on tube lid, heat block with heated lid or PCR machine is recommended for overnight incubation at 65 °C.

10. The ChIP and Input DNA can be diluted 1:5 to 1:10 for Q-PCR. Four microliters of diluted DNA can be used for each Q-PCR.