TITLE: Intact and clean nuclei isolation from wheat meristems
AUTHORS: Isabel Faci, Maximillian RW Jones, Cristobal Uauy

[DESCRIPTION]
This protocol describes how to perform nuclei isolation from wheat meristem tissues (spikes and carpels/ovaries), specifically from flash-frozen tissues. With this protocol, we obtain intact and clean nuclei for downstream applications.

[BEFORE_START]
NPBi can be prepared in advance and stored for up to 3 months in the fridge. 
It is highly recommended to read the full protocol before doing it.  

[STEPS]
SECTION: Tissue collection
2. Next, for each sample, label 2 mL Eppendorf tubes for collection and place two nitrogen-cooled metallic steel beads (previously cleaned with 0.5 M HCl and 70% ethanol, diameter 3 mm) in each tube. For each sample, we will pool a number of meristematic spikes/ovaries/spikelets. Depending on the stage/tissue, we will pool a different amount per sample (Table 1). Note that this is an indicative number for cultivar Chinese Spring. It is important not to use more tissue than indicated, as the filters may become blocked later in the protocol.  

 
 

 * Waddington scale stages:   

SECTION: Tissue collection
3. Once you have the number of plants that you need ready, prepare a stereo microscope to dissect and harvest the meristematic tissue. Make sure the area and the ophthalmic knives are clean (sterilise with 70% ethanol or RNase blitz, depending on what the nuclei isolation is for). 

SECTION: Tissue collection
4. Place the labelled 2 mL Eppendorf tubes with metallic beads on dry ice. 

SECTION: Tissue collection
5. Dissect the spikes/spikelets/ovaries/carpels under the microscope and place them in the pre-cooled collection tubes. For help with microdissections, take a look at this protocol:(dx.doi.org/10.17504/protocols.io.3byl49r2zgo5/v1). 

We usually follow this steps (considering all tubes being in dry ice, labelled, with metallic beads and lids opened): 

Clean surfaces and knives -> Dissect spike 1 -> Place spike 1 in sample A --> Clean surfaces and knives -> Dissect spike 2 -> Place spike 2 in sample B -> (...) 

We follow these steps during up to 30 minutes, or up to when the required amount based on Table 1 is reached. Then we close all the tubes and put them in liquid nitrogen. Then, if the tubes aren't completed yet, we put them back in dry ice, and repeat the same steps again, until the required amount based on Table 1 is reached. 

Note: Alternating which tube you deposit successive spikes/carpels/spikelets into will help to reduce variation between samples. Variation can arise due to the total collection time for a tube, the person dissecting, the time of day (many genes are regulated by the circadian clock), and many other factors!
SECTION: Tissue collection
6. You can continue the protocol on the same day or you can store the samples in -70 °C to -80 °C. 
SECTION: Tissue grinding
7. Next, grind the samples under cold conditions. We use the QIAGEN TissueLyser II, with the Adapter Set 2 x 24. Each adapter consists of a top plate, a bottom plate and a tube holder for holding 24 x 2 mL tubes. We pre-chill the 2 x top, 2 x bottom plates with dry ice for ~ 1 minute. We also fill the 2 x tube holders with dry ice, leaving free the spaces you will use for your 2 mL tubes. Once the adapter sets are cold, we quickly remove the dry ice from the 2 x bottom and 2 x top plates (but not from the tube holders), take the tubes from the liquid nitrogen and place them in the tube holders. Close them with the plates, and place them in the TissueLyser II, as quickly as possible. Grind them for 30 seconds at a frequency of 27.5 Hz (s¯¹). Put the tubes back in liquid nitrogen once finished. 

You can also grind with GenoGrinder, pre-cooling the tube holders with liquid nitrogen. 

Note: You can grind the samples in advance and keep them in -70 °C to -80 °C.
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
8. You can prepare in advance (up to 3 months earlier) the "incomplete Nuclei Purification Buffer" (NPBi) Stock (1 L) (Bajic et al., 2018).

Once all the reagents are added and well dissolved, and the pH adjusted to 7, filter sterilise with a syringe filter of 0.45 µm, 25 mm diameter. Then, degas under vacuum for 10 min. 
NPBi can be stored at 4 °C for up to 3 months. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
9. On the day, before starting the protocol, make sure you pre-chill the centrifuge to 4 °C. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
10. On the day, prepare the buffers (lysis, sucrose and complete Nuclei Purification Buffer (NPB)):

Prepare lysis buffer (contains β-mercaptoethanol, toxic: can be toxic if ingested, and fatal if inhaled or absorbed through the skin. Vapours can irritate the eyes, mucous membranes, and respiratory tract. Symptoms of inhalation exposure may include coughing, sore throat, and/or shortness of breath.) Prepare under the hood:

For 2 reps/samples, we make 5 mL (4 mL needed). Make lysis buffer and keep on ice. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
11. Prepare sucrose buffer contains B-mercaptoethanol (toxic), so should be prepared under the hood:

For 2 reps/samples, we make 5 mL (4 mL needed). Make lysis buffer and keep on ice.  

Label 2 x 5 mL tubes and place 2 mL of sucrose buffer in each ("sucrose cushion" tubes). Keep them on ice. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
12. Prepare Nuclei purification buffer (NPB) (Final volume needed for 2 reps/samples (1 mL per rep/sample) + error ~ 3 mL).

Make buffer and keep on ice.

ALL THE BUFFERS NEED TO BE PRE-CHILLED.  
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
13. Prepare 2 mL Eppendorf tubes with 100 µm filter and 70 µm filter (Figure 1).

 

SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
15. Make sure you have all the components ready to start:

- Ground tissue reps/samples in liquid nitrogen
- Centrifuge pre-chilled to 4 °C, with the right tube holders: 5 mL tubes
- Buffers (lysis, NPB) pre-chilled on ice
- "Sucrose cushion" tubes (5 mL size) pre-chilled on ice
- 100 µm filters in 2 mL tubes on ice
- 70 µm filters in 2 mL tubes on ice
- Cut-wide 1,000 µL tips 
- 5 mL pipettes
- Timer (20 secs)

Bear in mind, the next steps need to be done very quickly, but also gently. Stick to the timing, otherwise you will get fewer intact nuclei and more debris. Too much time or too much tissue results in lower quality. We recommend you memorise the steps and practise them beforehand. We also recommend that you do this between two people as each can do one sample at the same time. If only one person, we would recommend processing only one sample, and using a dummy sample to balance the centrifuge. 

SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
14. Prepare cut-wide tips (1 per rep/sample): cut 0.5 cm of 1,000 µL tips. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
16. 1) Take samples out of liquid nitrogen. Place them in a rack and open lids. Wait ~ 20 secs to add the lysis buffer (if you add it too fast, it will instantly freeze. Also, if there is too much tissue, it will more likely freeze). Try to add the lysis buffer at the exact same time in both samples (this is why having two people helps). Once added, put timer on for 20 s. 

2) In those 20 s, gently invert the tube a couple of times, carefully. When the timer is reaching 0, hold the Eppendorf tube with the 100 µm filter with your other hand. You will pour the liquid onto the filter, however the volume is 2 mL, so it is important that you hold the filter slightly higher than it would naturally do. Once the 20 s are over (and no later!), pour the sample onto the filter. If the filter blocks, don't force it. If you force it, you will get broken nuclei and debris. If you don't force it, you might lose yield but the nuclei will more likely be intact. (Most times the filter blocks due to having too much tissue, you can try with less if it blocks too much). 

3) Right after filtering with the 100 µm filter, remove the 100 um filter and do the exact same with the 70 µm filter (which should be in another 2 mL Eppendorf tube). Again, if the filter blocks, don't force it.

4) Remove the filter and pipette the 2 mL carefully with the cut-wide tip on top of the 2 mL sucrose cushions. Pipette it very gently, on the wall of the tube, so it falls into the cushion.

5) Centrifuge at 4 °C (2,200 g) for 20 minutes. 2200 x g, 4 °C 

SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
17. While the centrifuge runs, we prepare:
- the foil-covered DAPI tubes (to observe the nuclei under a fluorescence microscope): 0.5 µL 1 X DAPI per sample. Cover them in foil (DAPI is light-sensitive)
- Fluorescence microscope: Prepare ZEISS image software to take pictures under bright field and DAPI channels combined.
- Haemocytometer, cover slips
- Kimtech wipes and 70% ethanol

- Prepare 2 mL Eppendorf tubes with 40 µm filter and 20 µm filter, keep on ice (Figure 2)

 

SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
18. Once the centrifuge finishes, pick up the tubes carefully. Remove supernatant (the nuclei pellet is not visible, so just remove the supernatant slowly without touching the bottom of the tube). 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
19. Resuspend pellet in 1 mL of pre-chilled NPB by very gentle pipetting. 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
20. Pipette carefully and very slowly the 1 mL NPB with re-suspended nuclei into the filter walls of the 40 µm filter that is in a 2 mL Eppendorf tube (kept on ice). The filter will most likely block. Wait for 1 minute to see if it goes down by itself. If it doesn't, hit the filter ONCE very gently to help it go through. If it doesn't, wait 1 minute again. If after that 1 minute it is still blocked, don't do anything, just keep going with the volume that has gone trough. 

If you force it, you will get broken nuclei and debris. If you don't force it, you might lose yield but the nuclei will be more likely intact. (Most times the filter blocks due to having too much tissue, you can try with less if it blocks too much). 
SECTION: Nuclei isolation (we recommended to do two samples at a time with two people)
21. Repeat the exact same as step 22 with the 20 µm filter. 

The final volume will normally be ~ 0.9 - 1 mL, but it can also be lower if you have lost some sample because of filters blocking. If you want to calculate the amount of nuclei, you should record now how much approximate volume you have.

If you want to calculate the total number of nuclei, which is optional: Only 9.5 µL of each rep/sample will be used to quantify/visualise, keep the rest of the sample on ice as intact nuclei are more stable at cold temperatures.
SECTION: Visualise and quantify nuclei (optional)
22. Gently invert each sample to make sure that nuclei are well distributed within the sample. Then, add 9.5 µL of each rep/sample to the already prepared foil-covered tubes with 0.5 µL 1X DAPI. Mix by gentle pipetting. 
SECTION: Visualise and quantify nuclei (optional)
23. Place the slide coverslip on top of both chambers of the Bürker hemocytometer. Load 10 µl of your DAPI stained sample into one of the counting chambers with a 10 µl pipette. Capillary effect ensures even distribution of the suspension within the chamber. Place the haemocytometer under the microscope. The haemocytometer has 9 x 1 mm² squares. We often count DAPI stained (blue) intact (more or less round, with no membrane ruptures) nuclei in 3 random squares (out of 9), and calculate the total. 

Example: 

 

This is how we would then calculate the nuclei in this example:

Considering the volume of whole haemocyometer square: (9 mm2 x 0.1 mm depth = 0.9 mm3 = 0.0009 mL)
Volume of our sample is 0.9 mL. (Number here depends on the volume you observed in step 21)
Count (intact DAPI stained) nuclei in 3 yellow squares: 8 + 6 + 8 = 22 nuclei
Calculate nuclei in whole haemocytometer square. We counted 3/9 squares, so need to multiply by 3 to get an estimate for the whole haemocytometer chamber: 22 x 3 = 66 nuclei
Calculate total nuclei in 0.9 mL of sample:
0.9 mL total / 0.0009 mL haemocytometer square = 1,000
66 nuclei in haemocytometer x 1,000 = 66,000 nuclei in total

Note: To count nuclei, we often look closely into each of the yellow squares: Each of these contain 16 mini-squares. We take pictures in bright field and DAPI combined of each of the 16 mini-squares, to make sure we identify well the intact nuclei. The shape is well captured with bright field (rounded and intact), and the DAPI in the nuclei will be stronger than in other tissues. This is why is important to adjust well the microscope beforehand. 

Examples of real samples that can be improved (Figure 4): 


 


This is how a better sample (clean with intact nuclei) might look (Figure 5):

 

To visualise the next sample, clean with Kimtech wipes and ethanol, and load your next stained sample. 

Note: The nuclei will start bursting after roughly 15 minutes, especially if not kept on ice and if physical impact applied. We recommend you use them immediately after this protocol. This is also why we recommend doing few samples at a time, to be able to do all the steps faster and more gently. 

SECTION: Tissue collection
1. First, gather all the materials and prepare the necessary buffers described in the Materials section.