Preparation of single nuclei suspension from adult Drosophila abdominal cuticle

Preparation of single nuclei suspension from adult Drosophila abdominal cuticle

1.     Clean the SW 55 Ti rotor tubes 

2.     Start precooling the centrifuge and the rotor

3.     Prepare Lysis buffer 1 (4 ml)

4 ml Nuclei PURE lysis buffer (Provided in Sigma NUC-201 Nuclei isolation kit)

4 micro lit 1M DTT freshly thawed

40 micro lit 10% Triton-X (Provided in NUC-201 Kit)

5.     Sucrose cushion solution (For 1.5 M cushion)

To 6 ml of 2M sucrose cushion solution (NUC-201 Kit) add

2 ml of sucrose cushion buffer. Mix well.

6.     Dissect samples (40 adult male abdomens) in 1x relaxing buffer

Forty adult male abdomens were quickly dissected in ice cold relaxing buffer (1xPBS, 10 mM EGTA and 10 mM MgCl₂) by cutting off the last 2nd and abdominal segment with micro-scissors and hollowing out the abdomen by removing the intestines and male reproductive organs. The dissected tissue was then roughly chopped with micro-scissors (4-6 pieces per abdomen).

7.     Transfer chopped tissue samples to a 2 ml dounce homogenizer and pipette out all RB

8.     Add 0.3 ml of lysis buffer and homogenize sample by douncing 8 times (tight dounce)

9.     Add another 1 ml of lysis buffer and dounce ~15 times (tight dounce)

10.  Filter through the 40 micron sieve (Flowmi™ Cell Strainer for 1000µl Pipette Tips)

11.  Measure volume and make it up to 1.3 ml

12.  Transfer lysate to a new tube with 2.34 ml 1.5 M sucrose cushion solution, mix gently and set on ice

13.  Add 1.5 ml of 1.5 M sucrose cushion solution to prechilled centrifuge tubes (5ml tubes, Beckman)

14.  Layer on top the lysate sample carefully

15.  Repeat steps 11-14 with a mock lysis buffer to be used for balancing the rotor

16.  Place them in a prechilled SW 55 Ti rotor. Position 1 and 4

17.  Place the rotor in the prechilled centrifuge and spin at 30,000 g (17700 RPM) for 45 minutes at 4 C

18.  Remove the samples from the centrifuge

19.  Fat droplets will be visible on the top of the tube. Use a vacuum aspirator to quickly aspirate the top layer and the sucrose cushion all the way down to the lower rim of the tube (don’t go too close to the bottom, less the better). Make sure to go around the walls of the tube with the aspirator on the top of the tube. But stay at the center and aspirate slowly as you get closer to the bottom rim.

20.  Remove the remaining sucrose cushion with a 1 ml pipette tip without touching the bottom

21.  Add 400 micro lit of 10X recommended (1x PBS + 1% BSA + 0.2 units/ml of RNAse inhibitor) nuclei storage solution . 

22.  Collect the nuclei by centrifugation at 2500 RPM (sw55Ti) for 6 minutes

23.  Carefully pipette out the supernatant without touching the bottom.

24.  Resuspend the nuclei in 100 micro lit of nuclei storage solution

25.  Triturate 5-10 times

26.  Add 10 micro lit of the nuclei to storage buffer containing 2X DAPI. Mix by vortexing gently and use 10 micro lit to count nuclei

Count nuclei at every step to make sure nuclei are not lost at any step. The centrifuge speeds may vary a little from machine to machine and can significantly affect the final yield. Also make sure after homogenization that the nuclei are free and are not stuck to cell junk. Optimize douncing accordingly.

Later on we went for FACS based sorting of nuclei which was much faster and gave much cleaner nuclei even for adipose tissue samples. I would definitely recommend exploring that option too.

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