Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq

Scott Lindsay-Hewett

Published: 2022-03-19 DOI: 10.17504/protocols.io.kqdg3p2kel25/v1

Abstract

This protocol describes the isolation of nuclei from snap-frozen human placental tissue for bulk ATACseq. It is a modified version of a protocol (SCBL Protocols - 10x Multiome Nuclei Isolation) supplied by the Jackson Laboratory (JAX), which is itself modified from Sigma Aldrich's Nuclei Isolation Kit: Nuclei EZ Prep protocol, and 10x Genomics' Demonstrated Protocol – Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing. It is recommended to work with no more than four samples at a time so that all samples can be processed in a timely manner.

Steps

Preparation

Pre-chill microfuge to 4°C.

Prepare bucket of ice, and chill metal plate (allows for a flat surface to place Petri dish on top of - to keep tissue cool while cutting).

Dispense 200µL into microfuge tube and place on ice

Thaw 110µL aliquot of 100millimolar (mM) and place on ice.

Note

Prior to starting this protocol, prepare and freeze 110µL aliquots of 100millimolar (mM) from stock solution.

Thaw 500µL aliquot of tagmentation buffer and place on ice.

Note

Prior to starting this protocol, prepare 10mL tagmentation buffer as follows:330µL 220µL 367µL 1.6mL 7.483mL Freeze 500µL aliquots of tagmentation buffer.

Safety information

Take care when handling dimethylformamide. Wear appropriate personal protective equipment and work in a fume hood. Dispose of waste appropriately.

Pull snap-frozen placenta samples (~150-200 mg each) from -80 °C and place on dry ice until ready to process.

Note

This protocol describes nuclei isolation from 4 snap-frozen samples at a time. It is helpful to fill out a checklist (see below for example) to keep track of where each sample is in the protocol as you work through the various steps. Samples are staggered in such a way that the first sample is one step ahead of the second sample, which is one step ahead of the third sample, and so on.

ATACseq_sample_tracker.pdf

Label 4 microfuge tubes (the ones that are supplied with the disposable pestles) with sample names and pre-chill on ice.

Get the following materials ready; you won't have time to do this in between steps:

4 x #22 disposable scalpels

4 x 60mm Petri dishes

4 x disposable pestles/tubes, along with cordless motor

4 x disposable tweezers

4 x 40um filters

4 x 20um filters

4 x BioRad counting slides

0.4% Trypan Blue stain

200ul wide bore filter tips

1000ul wide bore filter tips

Make fresh buffers

Prepare 1mL as detailed below, and keep on ice:

988µL

2µL

10µL

Note

Check carefully that there is no precipitate in the 5% digitonin stock solution. If a white precipitate is present, heat at 94°C 0h 5m 0s. Re-dissolved digitonin is good for 1 week at room temperature.

10.

Prepare 8.5mL as detailed below, and keep on ice:

85µL

17µL

25.5µL

850µL

85µL

7352.5µL

Note

Dispense BSA solution in a laminar flow hood to maintain sterility of the stock solution.

Nuclei isolation

11.

Place a sterile Petri dish onto the chilled metal plate sitting on ice. Move tube containing first placenta sample from dry ice to wet ice for ~30 seconds, then tap slightly-thawed tissue into pre-chilled Petri dish.

12.

Cut tissue into small pieces (~ 2 to 3mm in size) using a fresh disposable #22 scalpel. Use disposable tweezers to hold tissue in place while cutting. Transfer tissue into labeled pre-chilled microfuge tube.

Safety information

Take care when cutting. If tissue is too hard and/or too much pressure is applied with the scalpel, tissue can fly out of the Petri dish. Cut cautiously and slowly to begin with - it is sometimes helpful to rock the blade from side to side to gradually split the frozen tissue. As the tissue thaws it gets a little easier to cut, but if it is allowed to thaw too much then it gets more difficult.

13.

Add 125µL and grind tissue with a disposable pestle attached to a cordless motor.

Note

It should only take a few 5 second pulses to achieve a "smoothie-like" consistency. Do not over-grind.

14.

Place ground-up tissue on ice for 0h 5m 0s. During this time you can start step 11 for the second placenta sample.

15.

Centrifuge sample 500x g,4°C. During this time you can start step 11 for the third placenta sample.

16.

Remove and discard supernatant, and resuspend pellet gently in 100µL using 200ul wide bore filter tips. Incubate on ice for 0h 5m 0s. During this time you can start step 11 for the fourth placenta sample.

17.

Using a 1000ul wide bore filter tip, add 500µL, mix gently, and centrifuge 500x g,4°C.

18.

Remove and discard supernatant and repeat wash twice, saving the last suspension on ice until all samples reach this step.

19.

Filter each sample though a 40um filter into a fresh microfuge tube. To maximize recovery of nuclei, wash old microfuge tube with 200µL, and pass through the same 40um filter.

20.

Repeat previous step, this time with a 20um filter. This filter will remove debris that wasn't removed in the previous step.

21.

Centrifuge 500x g,4°C.

22.

Remove and discard supernatant carefully, and gently resuspend pellet in 50µL using 200ul wide bore filter tip. Clumps of nuclei should break up easily. Place resuspended nuclei on ice.

23.

Take 5µL aliquot of resuspended nuclei; add 20µL and 25µL. Flick to mix, then load 10µL to both sides of a BioRad counting slide. Determine nuclei concentration using an automated cell counter.

Note

Nuclei should stain light blue; not too dark as that would indicate damage to the nuclear membrane. Nuclei should not be clumped together, and there should be little debris.

24.

Based on nuclei concentration, prepare 10µL of a 5,000 nuclei/ul solution (50,000 total nuclei), using tagmentation buffer for dilution. Proceed immediately to tagmentation and library preparation.

Note

It helps to use a pre-formulated Excel spreadsheet to quickly determine the volume of nuclei and tagmentation buffer to use. Several intermediate dilutions are often required to ensure that pipetting volumes are sensible.

Post-steps

25.

Centrifuge remaining nuclei from step 22 500x g,4°C. Remove and discard supernatant, and gently resuspend nuclei in 50µL using 200ul wide bore filter tips, making sure to break up clumps of nuclei. Store nuclei at -80°C for up to several months.

Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq

Abstract

Steps

Preparation

Make fresh buffers

Nuclei isolation

Post-steps

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