Protocol for nuclei isolation from fresh and frozen tissues for parallel snRNA-Seq and snATAC-Seq on 10x ChromiumTM platform using the same nuclei preparation (UPDATED version)
Luciano G Martelotto
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Abstract
This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas/paragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Before start
Attachments
Steps
Nuclei Prep and snRNA-Seq
Mince/chop tissue with a razor blade to small pieces. The tissue may be as small as 2 — 3 grains of (cooked) rice so long it can accommodate the sorting of sufficient nuclei for both snRNA-Seq and snATAC-Seq.
Add 300µL — 500µL of chilled Nuclei EZ Lysis Buffer (+ RNAse inhibitor) to the tissue in 1.5 ml DNA LoBind tube (for small pieces use 300µL).
Homogenise the sample using a douncer/pestle (gently stroking ~ 10 — 15 times).
Add more lysis buffer to 1mL, mix gently (bore tips preferred) and incubate 4On ice for at least 0h 5m 0s.
Filter homogenate using a 70 μm-strainer mesh to fit a 15 ml Falcon tube (e.g. pluriStrainer Mini 70 μm, Cell Strainer. My preferred one is 70-um Flowmi® Cell Strainer, in which case you would collect directly in 1.5 mL DNA LoBind tube).
Equipment
| Value | Label |
|---|---|
| pluriStrainer Mini 70 µm (Cell Strainer) | NAME |
| Cell Strainer | TYPE |
| pluriSelect | BRAND |
| 43-10070-40 | SKU |
Collect flow through in a 15 ml Falcon tube and transfer volume back into a new 1.5 ml DNA LoBind tube.
Centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C.
Remove supernatant leaving behind ~ 50µL.
Add 1mL of Nuclei Wash and Resuspension Buffer and gently resuspend the pellet (~1-2 pippete strokes).
Centrifuge at 500x g for 0h 5m 0s at 4°C.
Remove supernatant leaving behind ~ 50µL.
Add 1mL of Nuclei Wash and Resuspension Buffer. DO NOT resuspend the pellet.
Centrifuge at 500x g for 0h 5m 0s at 4°C.
Resuspend the nuclei in 200µL - 400µL Nuclei Wash and Resuspension Buffer supplemented with DAPI/7-AAD/DRAQ-7.
Collect all nuclei by washing off nuclei from the wall of centrifuge DNA LoBind tube.
Filter nuclei (at least once) with a 40-μm cell strainer (e.g. Falcon® Round-Bottom Tubes with Cell Strainer or Flowmi® Cell Strainer or 40-um Flowmi® Cell Strainer) before sorting.
Visually inspect nuclei integrity under a microscope and (optionally) count the number of nuclei with a cell counter (Countess II FL Automated Cell Counter) or hematocytometer.
Prior sorting, you may want to dilute sample to have ~ 150 — 200 events/second to get better defined peaks in cytometric analysis.
Perform cytometric analysis. Identify single nuclei and sub-populations based on DNA content, gate and sort directly into a round-bottom 96-well plate well containing the respective RT Buffer prepared without the RT Enzyme.
Proceed immediately with the 10x Genomics Single Cell 3' v3 or 5' protocol (Standard or NextGEM), minimising the time between nuclei preparation/sorting and chip loading.
Add the corresponding volume of RT Enzyme (depending on the kit, 10µL for 5', and 8.3µL for 3' v3) to the sorted nuclei in RT buffer.
Mix well but gently and load chip as per the Single Cell 3' v3 Reagents User Guide or Single Cell V(D)J 5' Reagents User Guide.
snATAC-Seq
Sort as many nuclei as possible into a round-bottom 96-well plate well containing 50µL of ice-cold ATAC Wash Buffer-Dig.
Transfer to 0.2 ml PCR tube (LoBind!).
Add 200µL of chilled ice-cold ATAC Wash Buffer-Dig.
Transfer any remanent nuclei to the 0.2 ml LoBind PCR tube (>/~250µL ).
Centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C.
Carefully remove the supernatant leaving behind ~ — . Do not disturb the pellet. 10µL — 15µL. Do not disturb the pellet.
Gently add 200µL ice-cold Diluted Nuclei Buffer.
Centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C.
Repeat Steps 28-30
Gently add 200µL ice-cold Diluted Nuclei Buffer.
Centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C.
Remove the supernatant in two steps , that is, remove a larger volume first using a P100/P200 pipette (~200µL) and then as much volume as possible with a P20 pipette leaving behind ~ — 7µL — . Do not disturb the pellet. 10µL. Do not disturb the pellet.
Resuspend nuclei in the ~ 7µL — 10µL of ice-cold Diluted Nuclei Buffer left behind, carefully washing walls of the tube to ensure all the nuclei are in solution.
Take 1µL — 2µL and dilute 1:5 with Diluted Nuclei Buffer.
Mix 1:1 with Trypan Blue and count the number of nuclei with a cell counter (Countess II FL Automated Cell Counter) or hematocytometer (the counting is to have an idea of how many nuclei to expect based on the recovery factor).
Use same slide to inspect under the microscope.
Take 5µL of nuclei in Diluted Nuclei Buffer and proceed directly to Chromium Single Cell ATAC Reagent Kits protocol (CG000168 Rev A). The volume added to the Transposition reaction will vary; for low input samples we usually use of the nuclei prep above 5µL of the nuclei prep above (see note in Step 31).
Alternatively, follow recommendations of the User Guide to estimate volume of nuclei to add to recover a determined targeted nuclei recovery (Page 20, CG000168 Rev A).
To estimate the Number of Recovered Nuclei, do the following calculation:
[Nuclei Concentration (from step 35) x Volume of Nuclei (up to 5 μl)] / 1.53 (recovery efficiency factor)
