CN105200004A - Separation and purification method of crosslinked tetrahymena micronucleus - Google Patents

Abstract

The invention relates to a method for separating and purifying Tetrahymena cells after cross-linking. Specifically, after cross-linking Tetrahymena cells with a cross-linking agent such as formaldehyde, the cells are lysed by a chemical lysis method, and finally separated by centrifugation. The small nucleus of Tetrahymena. It provides feasibility for Hi-C and related research in Tetrahymena thermophila.

Description

Method for separation and purification of Tetrahymena small nucleus after crosslinking

technical field

The invention belongs to the field of biological technology, and in particular relates to a separation method for purifying cross-linked Tetrahymena, especially the small nucleus of the thermophilic Tetrahymena, especially the small nucleus of the crescent stage.

Background technique

The existing method for separating the large and small nuclei of Tetrahymena is generally to separate the large and small nuclei by means of differential centrifugation or density gradient sedimentation after mechanical crushing. It is mainly operated on living cells, the operation time is long and it is not suitable for the separation of crescent nuclei, because the crescent nuclei are extremely extended from the original round nuclei to become linear nuclei, which are easy to break when mechanically broken. broken. In addition, because the cross-linked cells such as formaldehyde are difficult to mechanically disrupt, the current experimental method is not suitable for the cross-linked cells.

Tetrahymena thermophila is a good research model, however, the cross-linked cells cannot be effectively lysed and small nuclei are isolated, and it is difficult to obtain better crescent small nuclei. It makes it difficult to effectively carry out related experiments (ChIP, Hi-C) targeting small nuclei. In order to better study issues such as the transient recovery of transcriptional activity of small nuclei in zygote reproduction, it is necessary to establish a method that can be used to isolate small nuclei from Tetrahymena thermophila after cross-linking.

Contents of the invention

The present invention is mainly aimed at the separation of small nuclei of Tetrahymena thermophila after cross-linking, and is especially suitable for the separation of small nuclei in a special period (crescent phase), and can be used for subsequent Hi-C, ChIP-seq and other experiments. As far as the Hi-C experiment is concerned, it is a technique that can be used to study chromatin conformation and interaction at the genome-wide level, and has received extensive attention in recent years. Tetrahymena has two nuclei, and there are multiple copies of chromosomes in the large nucleus, which is not suitable for subsequent analysis and research of Hi-C experiments, so it is necessary to separate and purify its small nucleus for subsequent experimental research. In addition, the crescent of Tetrahymena The small nuclei are a good material to study the conformational changes of chromatin. Therefore, the present invention provides feasibility for Hi-C in Tetrahymena thermophila and related research.

In the present invention, after cross-linking the Tetrahymena thermophila cells with a cross-linking agent such as formaldehyde, the cells are lysed by a chemical lysis method, and finally the small nuclei of the Tetrahymena thermophila are separated by a centrifugation method. The technical scheme of the present invention includes: 1) using a cross-linking agent such as formaldehyde to carry out cross-linking treatment on Tetrahymena thermophila cells; 2) treating Tetrahymena thermophila cells with a chemical lysate to make the cell membrane easily broken; 3) using SDS lysing the cells; 4) removing the large nuclei by repeated centrifugation to finally obtain the small nuclei of Tetrahymena thermophila.

The technology of the present invention fixes cells with a cross-linking agent, uses a chemical method to lyse the cells, and then separates large nuclei and small nuclei. Not only ordinary circular nuclei can be obtained, but also linear nuclei in the crescent stage of sexual reproduction of Tetrahymena thermophila can be isolated.

Specifically, the present invention includes the following items:

1. A method of isolating the small nucleus of Tetrahymena, said method comprising:

1) using a cross-linking agent to carry out cross-linking treatment on Tetrahymena cells;

2) use SDS to lyse the cells;

3) Remove large nuclei by centrifugation to obtain small nuclei.

2. The method according to 1, further comprising the step of treating Tetrahymena cells with a cell lysate before step 2).

3. The method of claim 1, wherein the Tetrahymena is Tetrahymena thermophila.

4. The method according to 1, wherein the crosslinking agent is formaldehyde.

5. The method according to 1, wherein the cell lysate has the following composition: 20mM Tris-HCl pH8.0, 100mMNaCl, 20mMKCl, 1.5mMMgCl2, 1% NP-40.

6. The method according to 5, wherein the cell lysate further comprises a cocktail of protease inhibitors.

7. The method according to 1, wherein the nuclei are crescent nuclei.

8. The method according to 1, wherein the lysing step in step 3) requires dispersing the cells into single cells.

9. The method according to 1, wherein the lysis time in step 3) is 10-15 minutes, preferably 12 minutes.

10. The method according to 1, wherein the centrifugation step in step 4) is repeated several times.

The present invention has the following advantages and positive effects:

The existing separation methods are mainly used for the separation of large and small nuclei in living cells of Tetrahymena thermophila, and are not suitable for the separation of small nuclei in the crescent stage. The method we established for the isolation of small nuclei from Tetrahymena thermophila is mainly for Hi-C, ChIP and other experiments, which require the cells to be fixed with formaldehyde. In addition, our separation method can not only isolate general round nuclei, but also can be used for the isolation of special crescent nuclei. The establishment of this method allows Hi-C, ChIP-seq and other experiments targeting the small nucleus of Tetrahymena thermophila to be carried out. This provides convenience for the genome function research of Tetrahymena thermophila small nucleus.

Description of drawings

Figure 1. Segregation of crescent nuclei during conjugation of Tetrahymena thermophila.

Detailed ways

The present invention is realized by the following means.

I. Sample collection and storage

1. Take Tetrahymena in crescent stage 3 hours after conjugation, with a density of about 2.5x10 ⁵ cells/ml, add 37% formaldehyde to a final concentration of 1%, mix well, and leave at room temperature for 10 minutes.

2. Add 2.5M glycine to a final concentration of 125mM to neutralize formaldehyde, mix well, place at room temperature for 5 minutes, and place on ice for 15 minutes.

3. Centrifuge at 1500g for 2 minutes at 4°C, remove the supernatant, and wash once with 10mM Tris-HCl. Centrifuge again to remove supernatant.

4. Transfer to a 1.5ml centrifuge tube, about 2x107 cells per tube, centrifuge at 2000g, ⁴ degrees Celsius for several times, and remove residual liquid as much as possible. Samples can be stored at -80°C or used directly for subsequent experiments.

II. Cell Lysis and Isolation of Crescent Nuclei

1. Take the Tetrahymena samples (2x107 cells/tube) preserved at -80 degrees Celsius, and resuspend each tube of samples in 1ml of cell lysate (20mM Tris-HClpH8.0, ^100mMNaCl , 20mMKCl, 1.5mMMgCl2, 1 %NP-40, add 1x protease inhibitor mixture (Roche product number: Cat. No. 11873580001)) before use, rotate and lyse at 4 degrees Celsius for 1 hour.

2. Centrifuge at 2000g at 4°C to remove the supernatant, wash once with 500ul pre-cooled cell lysate, and remove the lysate after centrifugation.

5. Resuspend the cells with 400ul of 0.5% SDS, try to disperse the cells into single cells during resuspension, avoid excessive cell aggregation, and shake at 62 degrees Celsius for about 12 minutes.

6. Add 200ul 10% TritonX-100 and 200ul water to neutralize SDS, and mix well.

7. Centrifuge at 780g at 4°C for 12 minutes, carefully transfer the supernatant to a new centrifuge tube (small nuclei), centrifuge again at 780g at 4°C for 12 minutes, transfer the supernatant to a new centrifuge tube (remove residual large nuclei, can Repeat several times).

10. Take a part for DAPI staining to identify the effect of small nucleus separation (as shown in Figure 1). The remaining sample was centrifuged at 20,000g at 4°C for 12 minutes, the supernatant was discarded, and the pellet was small nuclei.

In the analysis results (Figure 1), Lysis is the lysis effect diagram after cell lysis, including linear small crescent nuclei, circular small nuclei, and circular large nuclei. MIC-1, 2, and 3 are the renderings of the final purified small nuclei, which are basically circular and linear small nuclei, with little contamination from large nuclei. Thus, the present invention proved to be very effective for isolating small nuclei, especially crescent small nuclei.

Claims (10)

1. be separated a method for the small nut of thermophilas, described method comprises:

1) linking agent is used to carry out crosslinking Treatment to thermophilas cell;

2) SDS lysing cell is used;

3) centrifugal removing macronucleus, obtains small nut.

2. method according to claim 1, described method is also included in step 2) before by the step of cell pyrolysis liquid process thermophilas cell.

3. method according to claim 1, wherein said thermophilas is tetrahymena thermophila.

4. method according to claim 1, wherein said linking agent is formaldehyde.

5. method according to claim 1, wherein said cell pyrolysis liquid has following composition: 20mMTris-HClpH8.0,100mMNaCl, 20mMKCl, 1.5mMMgCl2,1%NP-40.

6. method according to claim 5, wherein said cell pyrolysis liquid also comprises protease inhibitor cocktail.

7. method according to claim 1, wherein said small nut is crescent phase small nut.

8. method according to claim 1, wherein step 3) in cleavage step need cell dispersal to become unicellular.

9. method according to claim 1, wherein step 3) in pyrolysis time be 10-15 minute, be preferably 12 minutes.

10. method according to claim 1, wherein repeating step 4) in centrifugation step for several times.