Nov 21, 2022
Nuclei Isolation and Sorting from Frozen Human Temporal Cortex
- Clemens Scherzer1,2
- 1Brigham and Women's Hospital;
- 2Harvard Medical School
Protocol Citation: Clemens Scherzer 2022. Nuclei Isolation and Sorting from Frozen Human Temporal Cortex. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk4k55g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2020
Last Modified: May 31, 2024
Protocol Integer ID: 43801
Keywords: nuclei isolation, frozen human temporal cortex, ASAPCRN
Abstract
This protocol is about nuclei isolation and sorting from frozen human temporal cortex.
Attachments
Materials
1. Dounce tissue grinder setSigma AldrichCatalog #D8938-1SET
2. Nuclei Isolation Kit: Nuclei PURE PrepSigma AldrichCatalog #NUC201-1KT
3. DTTSigma AldrichCatalog #43816-10ML
4. DPBS with no calcium and magnesiumThermo Fisher ScientificCatalog #14190-144
5. UltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616
6. Recombinant RNase InhibitorClontechCatalog #2313B
7. 70 μm Sterile Cell StrainerFisher ScientificCatalog #22363548
8. Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
9. ART™ Wide Bore Filtered Pipette TipsThermo Fisher ScientificCatalog #2079G
Buffer (For 2 samples):
- Lysis buffer (LB):
8 mL Nuclei PURE Lysis buffer
8 µL DTT
80 µL 10% Triton X-100
2. Nuclei wash and resuspension buffer (NWRB):
14.8 mL DPBS
150 µL BSA
75 µL RNase Inhibitor (40 U/ml)
3. Nuclei wash and resuspension buffer with DAPI (NWRBD):
5.5 mL NWRB
11 µL DAPI (5 mg/ μl)
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Method
Method
40m
40m
Cut 100 mg - 200 mg frozen temporal cortex tissue into small pieces on dry ice.
Put cut tissue into a microcentrifuge tube (pre-cooled on dry ice), weigh in a balance.
Transfer tissue to a glass dounce tissue grinder on regular ice.
Add 2 mL LB to the tissue grinder.
Homogenize tissue with pastel A 10 times and pastel B 10 times.
Transfer the homogenate into a 2 ml microcentrifuge tube.
Incubate On ice for 00:05:00 , mix with Wide bore tips once during the incubation.
5m
Filter the homogenate with a 70 μmSstrainer.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Remove the supernatant and resuspend the pellet in 1.5 mL LB with Wide bore tips.
Incubate On ice for 00:05:00 .
5m
Centrifuge at 500 x g, 4°C, 00:05:00 .
Remove the supernatant and add 500 µL NWRB to the pellet without resuspension.
Incubate On ice for 00:05:00 .
5m
Add 1.0 mL additional NWRB to the tube and resuspend the pellet.
Centrifuge at 500 x g, 4°C, 00:05:00 .
Remove the supernatant and resuspend the pellet in 1.8 mL NWRB .
Pipette 200 µL nuclei from original tube (T1) into a new tube (T2).
Centrifuge both T1 and T2 at 500 x g, 4°C, 00:05:00 .
Remove supernatant in T1 and resuspend pellet with 500 µL NWRBD .
Remove supernatant in T2 and resuspend pellet with 65 µL NWRB .
Filter nuclei in T1 and T2 with 35 μm strainer respectively.
Check nuclei under microscope and count the number with cell counter (Optional).
Add 2 mL NWRBD to T1, mix by pipetting.
Add 240 µL NWRB to T2, mix by pipetting.
Use T2 as a negative control for sorting.
Sort nuclei from P3 gate into a 1.5 ml microfuge tube preloading 15 µL NWRB .
Collect 90,000 events from P3 gate, the final volume is about 40 µL - 60 µL .
Centrifuge at 500 x g, 4°C, 00:05:00 .
Remove supernatant and resuspend pellet with 15 µL NWRB .
Use 8.4 µL of nuclei (50,400 events) for 10x GEM generation and barcoding following manufacture´s protocol.