Mar 09, 2020

Public workspaceProtocol 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) V.4

DOI
dx.doi.org/10.17504/protocols.io.bdeni3de
  • 1University of Adelaide
  • Human Cell Atlas Method Development Community
  • Single Cell Core, Harvard Medical School
Luciano G Martelotto
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bdeni3de
Protocol Citation: Luciano G Martelotto 2020. 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). protocols.io https://dx.doi.org/10.17504/protocols.io.bdeni3de
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 in our group and it is working. Please get in touch if you need help. Version 4 is the latest version with some improvements.
Created: March 09, 2020
Last Modified: March 09, 2020
Protocol Integer ID: 33966
Keywords: nuclei isolation, snRNA-Seq, snATAC-Seq, 10x Chromium platform
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).
Attachments
Icon for Protocol_draft10.pdf
Protocol_draft10.pdf
1.1MB
Guidelines
Protocol Overview
Note
This protocol requires access to a cell sorter and familiarity with sorting cells/nuclei into 96-well plates.

Note
SUCCESS of this protocol heavily rely on SAMPLE QUALITY. Below are some of the steps I follow:

-For new sample types, and when possible (with minute samples is hard to do it) I evaluate the lysis efficacy by assessing under light or fluorescent microscope after Duration00:03:00 -Duration00:05:00 for single cell suspensions (cell lines) and Duration00:05:00 -Duration00:10:00 for tissues. This will ensure you avoid over- or under-lysis the cells.

-The cell lysis and washes are always carried out on wet-ice (i.e. Temperature4 °C ) and in the presence of RNAse inhibitor 0.2-0.5 U/uL.

-Centrifugation, in my hands, works well at Centrifigation500 x g, 4°C, 00:05:00 and this is enough for most of the tissues types I worked on. However, some optimization might be needed for specific tissues.

-I always use LoBind nucleases free tubes.



-I always inspect nuclei under microscope using Trypan Blue to give more contrast and also to count.

-When debris and clump are an issue and I solved this issue I use sorting as explained in the protocols.

-Nuclei sizes and shapes under microscope varies from sample to sample so shape or roundness is not the only feature to check. Also look for signs of disorganisation of chromatin, this is usually quite visible as if burst nucleus.

-In good quality nuclei nucleoli may be visible.

-I use Flowmi 40 μm filters before FACS sorting or before loading onto chip.

-For additional tips on sample prep please check https://support.10xgenomics.com/single-cell-gene-expression/sample-prep.


  1. Use a plastic pestle to mechanically homogenize tissue and release nuclei
  2. Separate the nuclei from debris using a cell sorter (if not, then see note at the end)
  3. Collect a specific number of nuclei in a 96-well plate containing 10x RT Buffer* or Wash Buffer**
  4. Immediately load the sample into a Single Cell Chip for processing according the Single Cell 3’ v3 Reagents User Guide or Single Cell V(D)J 5’ Reagents User Guide.
  5. In our hands the use of DAPI, 7-AAD and DRAQ-7 dyes show very little or no effect on ATAC data metrics.

*Consider the event overestimation of some sorters (Check step 19). Also, assume that nuclei recovery is ~ 57 %; use this to determine the number of nuclei to collect for each of your samples. [This value is derived from the Cell Suspension Volume Calculator Table in the Single Cell 3’ and v3 - Reagents User Guide or Single Cell V(D)J 5’ v1 Reagents User Guide]
**Assume that nuclei loss can be up to ~ 50 — 60 % due to loss during washing and counting; use this to determine the number of nuclei to collect for each of your samples.



Note
IMPORTANT NOTE:

If you are NOT sorting nuclei that is in Nuclei Wash and Resuspension Buffer directly into ATAC Wash Buffer-Dig, avoid resuspending pelleted nuclei that was in Nuclei Wash and Resuspension Buffer (PBS-based) directly in Diluted Nuclei Buffer or ATAC Wash Buffer-Dig as nuclei tend to clump.

Instead, you need to do a buffer exchange (from PBS to Tris) by adding at least 2x the volume of ATAC Wash Buffer-Dig [Tris-HCl 10 mM (pH 7.4), NaCl 10 mM, MgCl2 3 mM, BSA 1%, Tween-20 0.1 %, Digitonin 0.01%] to the nuclei that is in suspension in the Nuclei Wash and Resuspension Buffer (PBS-based).

Let equilibrate for Duration00:05:00 (buffer exchange). After this, pellet and do all washes in ATAC Wash Buffer-DIg before resuspending nuclei in Diluted Nuclei Buffer.


Below are examples of nuclei before and after sorting, bioanalyzer traces of 5’ nuclei cDNA and snATAC library as well as representative metrics obtained with Cell Ranger for this example.
Fig 1. Nuclei before sorting.


Fig 2. Nuclei after sorting.


Fig 3. cDNA and ATAC libary traces. Metrics obtained with Cell Ranger
Fig 4. Representative photos of how clean the nuclei looks after sorting and right before loaidng on the Chromium.
Fig 5. TapeStation traces of ATAC libraries.
Fig 6. No Dye: Representative ATAC QC metrics from CellRanger.
Fig 7. DAPI: Representative ATAC QC metrics from CellRanger.
Fig 8. SYTO RNASelect: Representative ATAC QC metrics from CellRanger.
Fig 9. PI: Representative ATAC QC metrics from CellRanger.
Fig 10. 7-AAD: Representative ATAC QC metrics from CellRanger.
Fig 1. DRAQ-7: Representative ATAC QC metrics from CellRanger. NOTE: this dye works very well, but for this particular experiment the sorter was a bit jittery and some bad quality nuclei were sorted, so we suspect the metrics were affected by it.

Materials
Required Buffers and Reagents

1. Nuclei EZ Lysis Buffer (Millipore Sigma) (chilled, Temperature4 °C )

2. Nuclei Wash and Resuspension Buffer (prepare chilled, Temperature4 °C )
1x PBS (No Ca2+/Mg2+)
Concentration1.0 % volume BSA
Concentration0.2 Mass Percent — Concentration0.5 Mass Percent RNase Inhibitor

3. Nuclei Wash and Resuspension Buffer with DNA binding dye (prepare chilled, Temperature4 °C )
1x PBS (No Ca2+/Mg2+)
Concentration1.0 % volume BSA
Concentration0.2 Mass Percent — Concentration0.5 Mass Percent RNase Inhibitor
Concentration10 Mass Percent DAPI or Concentration1 Mass Percent 7-AAD or Concentration3 micromolar (µM) DRAQ-7 (see notes)

4. RT Buffer* for Single Cell Gene Expression 3’ v3 reagents (DO NOT add RT Enzyme C)
RT Reagent: Amount20 µL
Template Switch Oligo: Amount3.1 µL
Reducing Agent B: Amount2.0 µL
H2O: (33.4 – X – Y) μl

5. RT Buffer* for Single Cell Immune Profiling 5’ reagents (DO NOT add RT Enzyme Mix B)
RT Reagent Mix: Amount50 µL
RT primer: Amount5.9 µL
Additive A: Amount2.4 µL
H2O: (31.7 – X – Y) μl

6. Diluted Nuclei Buffer (chilled, Temperature4 °C )
20x Nuclei Buffer (ATAC kit, part 2000153): Amount50 µL
H2O: Amount950 µL

7. ATAC Wash Buffer-Dig (chilled, Temperature4 °C )
Tris-HCl (Ph7.4 ): Concentration10 millimolar (mM)
NaCl: Concentration10 millimolar (mM)
MgCl2: Concentration3 millimolar (mM)
BSA: Concentration1.0 Mass Percent
Tween-20: Concentration0.1 Mass Percent
Digitonin: Concentration0.01 Mass Percent


* RT Buffer Notes
  • X (‘sorting volume’): In the cytometric analysis setup described in this protocol, each droplet is 1 nl. Example: 10,000 nuclei = 10,000 nl = 10 μl ‘sorting volume’.
  • Y (‘additional volume’): This accounts for any additional volume deposited by the flow cytometer nozzle. In the cytometric analysis setup described in this protocol (i.e. 70 μm nozzle) there is no additional volume deposited by the nozzle, so Y = 0. If in doubt, or to be on the safe side, just make Y = 5 — 10 μl.
  • The 1 nuclei/nl assumption was corroborated empirically by sorting 10,000 nuclei in ten wells containing 70 μl PBS and then measuring the final volume post sorting. It is highly recommended to determine X empirically as value may vary depending on different sorters/nozzle combinations. It is recommended to determine it at least once.
  • Always measure the volume after sorting and top up to 90 μl with PBS or H2O if required.
  • After adding the RT Enzyme Mix the final volume should be ~ 100 μl.
  • It is crucial to work as fast as possible. Do not leave nuclei sitting on ice for too long (e.g. 30’ is too long).
  • Reduce as much as possible the time from sorting-to-controller run, ideally keep it under 40’. The longer the time the higher the background will be.
  • The sorting and/or resuspension of nuclei in Diluted Nuclei Buffer is critical for optimal snATAC-Seq assay performance.
  • Use DNA LoBind tubes for all steps (when possible)!
  • Since this protocol does not use nor need Digitonin during the initial cell lysis, it is important to include Tween-20 and Digitonin 0.01% in the ATAC Wash Buffer-Dig as it helps in nucleus envelope permeabilisation. Note, Digitonin is optional but recommended.


Additional equipment required
  • Protector RNAse Inhibitor (Cat. Number: RNAINH-RO, Merck/Roche)
  • Nuclei EZ Lysis Buffer (sold as Nuclei Isolation Kit: Nuclei EZ Prep by Merck/Sigma, Cat. Number: NUC101)
  • 1.5 ml DNA LoBind Eppendorf tubes
  • 0.2 μl PCR-tubes/strips (LoBind)
  • 15 ml Falcon tubes
  • 70 μm-strainer mesh to fit a 15 mL Falcon tube (e.g. pluriStrainer Mini 70 um, Cell Strainer or Flowmi® Cell Strainer)
  • 40-μm cell strainer (e.g. Falcon® RoundBottom Tubes with Cell Strainer or Flowmi® Cell Strainer)
  • Round-bottom 96-well plate
  • Light or fluorescent microscope
  • FACS instrument (i.e. BD FACSAriaTM Fusion, SONY SH800S), ideally with 70 μm nozzle.
  • Thermocycler
  • TapeStation or Bioanalyzer plus consumables
  • Cell counter/hematocytometer
  • Douncer/pestle
  • Refrigerated centrifuge
  • Razor blades

























Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Note
All samples and reagents are kept TemperatureOn ice or at Temperature4 °C .




Nuclei Prep and snRNA-Seq
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.
Note
Note that nuclei yield largely depends on tissue cellularity.

Add Amount300 µL — Amount500 µL of chilled Nuclei EZ Lysis Buffer (+ RNAse inhibitor) to the tissue in 1.5 ml DNA LoBind tube (for small pieces use Amount300 µL ).
Homogenise the sample using a douncer/pestle (gently stroking ~ 10 — 15 times).
Note
For mincing the tissue, you may take the DNA LoBind tube out of the ice, however, be quick and return to ice.

Add more lysis buffer to Amount1 mL , mix gently (bore tips preferred) and incubate TemperatureOn ice for at least Duration00:05:00 .
Note
Lysis time of 5 min has been enough for most tissues we tested, but you may need some optimization.


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
pluriStrainer Mini 70 µm (Cell Strainer)
NAME
Cell Strainer
TYPE
pluriSelect
BRAND
43-10070-40
SKU
https://www.pluriselect.com/us/pluristrainer-mini-70-m-cell-strainer.html#size=27
LINK

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 Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Remove supernatant leaving behind ~ Amount50 µL .
Add Amount1 mL of Nuclei Wash and Resuspension Buffer and gently resuspend the pellet (~1-2 pippete strokes).
Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Remove supernatant leaving behind ~ Amount50 µL .
Add Amount1 mL of Nuclei Wash and Resuspension Buffer. DO NOT resuspend the pellet.

Note
Additional washes are possible but may incur in nuclei loss. If doing so, I recommend not to resuspend nuclei in between washes.

Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Resuspend the nuclei in Amount200 µL - Amount400 µL Nuclei Wash and Resuspension Buffer supplemented with DAPI/7-AAD/DRAQ-7.
Note
Resuspension volume can vary to achieve ~ 150 — 200 events/second during cyrtometric analysis (see step 18).




Collect all nuclei by washing off nuclei from the wall of centrifuge DNA LoBind tube.
Note

IMPORTANT: Protect from light from here forward.

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.
Note
IMPORTANT: for snRNA-Seq, we have seen that FACS sorters tend to overestimate the number of nuclei sorted in about >/= 40 % depending on instrument, so we usually sort 35 — 40 % more nuclei than aimed (e.g. for ~ 5000 nuclei recovery, you would need ~ 8,700 but we sort ~ 12,000 nuclei).

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, Amount10 µL for 5', and Amount8.3 µL 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
snATAC-Seq

Note
Ideally, perform the following steps while the RT reaction is running.
The protocol below assumes the nuclei input is low.

Sort as many nuclei as possible into a round-bottom 96-well plate well containing Amount50 µL of ice-cold ATAC Wash Buffer-Dig.
Note
DO NOT, I repeat DO NOT sort into Diluted Nuclei Buffer

Expected result
We have successfully sorted as little as ~7000 nuclei and recovered ~3500+ profiled nuclei (~50 % recovery as expected, 1.53 recovery efficiency factor).

Note
Note there will be significant nuclei loss during washes and nuclei counting, so you may want to make sure the washing steps are done carefully. Take into account this loss when deciding aimed nuclei. To reduce loss, follow the tips below (bold).


Transfer to 0.2 ml PCR tube (LoBind!).
Add Amount200 µL of chilled ice-cold ATAC Wash Buffer-Dig.
Note
For this step, Diluted Nuclei Buffer can also be used.




Transfer any remanent nuclei to the 0.2 ml LoBind PCR tube (>/~Amount250 µL ).

Centrifuge the nuclei at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Carefully remove the supernatant leaving behind ~Amount10 µL — Amount15 µL . Do not disturb the pellet.
Note
You may remove supernatant in 2-3 pipetting steps, namely, a large volume first (~Amount200 µL ) using a P200, followed by small volumes using a P20.



Gently add Amount200 µL ice-cold Diluted Nuclei Buffer.
Note
DO NOT resuspend nuclei.

Centrifuge the nuclei at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Repeat Steps 28-30
Gently add Amount200 µL ice-cold Diluted Nuclei Buffer.
Note
DO NOT resuspend nuclei.



Centrifuge the nuclei at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C .
Remove the supernatant in two steps, that is, remove a larger volume first using a P100/P200 pipette (~Amount200 µL ) and then as much volume as possible with a P20 pipette leaving behind ~ Amount7 µL — Amount10 µL . Do not disturb the pellet.



Note
Alternatively, if the nuclei are limited, you may resuspend the pellet in Amount5 µL of Diluted Nuclei Buffer and proceed directly onto Step 36. Do not count as it'll be innacurate anyways and you'll lose precious nuclei.


Resuspend nuclei in the ~ Amount7 µL — Amount10 µL of ice-cold Diluted Nuclei Buffer left behind, carefully washing walls of the tube to ensure all the nuclei are in solution.
Take Amount1 µL — Amount2 µL 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 Amount5 µL 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 Amount5 µL 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)