Mar 19, 2022
Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq
- 1University of California, San Diego
Protocol Citation: Scott Lindsay-Hewett 2022. Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p2kel25/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: March 17, 2022
Last Modified: March 19, 2022
Protocol Integer ID: 59544
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.
Materials
Equipment:
Benchtop microfuge with cooling function
Fume hood
Laminar flow hood
Cordless motor for disposable pestles (Z359971-1EA)
Metal plate
Automated cell counter
Heating block
Set of pipettes
Microscope
Supplies/reagents:
60mm Petri dishes (Fisher, cat # FB0875713A)
LoBind Eppendorf tubes (Eppendorf, cat # 022431021)
#22 sterile disposable scalpels (Fisher, cat # 22-079-714)
Trizma hydrochloride solution, 1M, pH 7.4 (Sigma, cat # T2194-100ML)
Magnesium chloride solution, 1M (Sigma, cat # M1028-100ML)
Nuclei Isolation Kit: Nuclei EZ Prep (Sigma, cat # NUC101-1KT)
Magnesium acetate tetrahydrate (Sigma, cat # M2545-250G) - use to prepare 300mM solution, filter sterilize
N,N-Dimethylformamide (Sigma, cat # 227056-100ML)
Trypan Blue solution, 0.4% (Sigma, cat # T8154-20ML)
Nuclease-free water
Bel-Art Disposable Pestles, polypropylene, With 1.5 mL Tubes, pack of 100 (Sigma, cat # BAF199230000-100EA)
Single-use tweezers, angled,blue,sterile (Thomas Scientific, cat # 19A00N163)
Dithiothreitol (DTT), 0.1M Solution (Fisher, cat # 707265ML)
Pluriselect pluriStrainer Mini 40 UM, 100/Bag (Fisher, cat # NC1469671)
Pluriselect PluriStrainer Mini 20 µm Cell Strainer, 100/Pk, Sterile (Fisher, cat # NC1423042)
Digitonin, 5% (Fisher, cat # BN2006)
Tween-20, 10%
NaCl (5 M), RNase-free (Fisher, cat # AM9760G)
Bovine Serum Albumin solution, 10% in DPBS (Sigma, cat # A1595-50ML)
200ul wide bore filter tips (Fisher, cat # 2069GPK)
1000ul wide bore filter tips (Fisher, cat # 2079GPK)
Cell Counting Slides for TC10™/TC20™ Cell Counter, Dual-Chamber (Bio-Rad, cat # 1450011)
Tris base (Fisher, cat # BP152-500) - use to prepare 1M solution; adjust to pH 7.8 using glacial acetic acid, filter sterilize
Potassium acetate (Sigma, cat # P5708-500G) - use to prepare 3M solution, filter sterilize
Bucket of ice
Filter tips
Preparation
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 200 µL Nuclei EZ Storage Buffer into microfuge tube and place on ice
Thaw 110 µL aliquot of 100 millimolar (mM) DTT and place on ice.
Note
Prior to starting this protocol, prepare and freeze 110 µL aliquots of 100 millimolar (mM) DTT from stock solution.
Thaw 500 µL aliquot of tagmentation buffer and place on ice.
Note
Prior to starting this protocol, prepare 10 mL tagmentation buffer as follows:
330 µL 1M Tris-acetate, pH 7.8
220 µL 3M Potassium acetate
367 µL 300mM Magnesium acetate
1.6 mL Dimethylformamide
7.483 mL Nuclease-free water
Freeze 500 µL 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
Make fresh buffers
Prepare 1 mL fresh lysis buffer as detailed below, and keep on ice:
988 µL EZ lysis buffer
2 µL 5% Digitonin
10 µL 100mM DTT
Note
Check carefully that there is no precipitate in the 5% digitonin stock solution. If a white precipitate is present, heat at 94 °C 00:05:00 . Re-dissolved digitonin is good for 1 week at room temperature.
Prepare 8.5 mL wash buffer as detailed below, and keep on ice:
85 µL 1M Tris-Cl, pH 7.4
17 µL 5M Sodium chloride
25.5 µL 1M Magnesium chloride
850 µL 10% Tween-20
85 µL 10% BSA
85 µL 100mM DTT
7352.5 µL Nuclease-free water
Note
Dispense BSA solution in a laminar flow hood to maintain sterility of the stock solution.
Nuclei isolation
Nuclei isolation
25m
25m
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.
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.
Add 125 µL lysis buffer 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.
Place ground-up tissue on ice for 00:05:00 . During this time you can start step 11 for the second placenta sample.
5m
Centrifuge sample 500 x g, 4°C, 00:05:00 . During this time you can start step 11 for the third placenta sample.
5m
Remove and discard supernatant, and resuspend pellet gently in 100 µL fresh lysis buffer using 200ul wide bore filter tips. Incubate on ice for 00:05:00 . During this time you can start step 11 for the fourth placenta sample.
5m
Using a 1000ul wide bore filter tip, add 500 µL wash buffer , mix gently, and centrifuge 500 x g, 4°C, 00:05:00 .
5m
Remove and discard supernatant and repeat wash twice, saving the last suspension on ice until all samples reach this step.
Filter each sample though a 40um filter into a fresh microfuge tube. To maximize recovery of nuclei, wash old microfuge tube with 200 µL fresh wash buffer , and pass through the same 40um filter.
Repeat previous step, this time with a 20um filter. This filter will remove debris that wasn't removed in the previous step.
Centrifuge 500 x g, 4°C, 00:05:00 .
5m
Remove and discard supernatant carefully, and gently resuspend pellet in 50 µL tagmentation buffer using 200ul wide bore filter tip. Clumps of nuclei should break up easily. Place resuspended nuclei on ice.
Take 5 µL aliquot of resuspended nuclei; add 20 µL tagmentation buffer and 25 µL 0.4% Trypan blue stain . Flick to mix, then load 10 µL 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.
Based on nuclei concentration, prepare 10 µL 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
Post-steps
5m
5m
Centrifuge remaining nuclei from step 22 500 x g, 4°C, 00:05:00 . Remove and discard supernatant, and gently resuspend nuclei in 50 µL Nuclei EZ Storage Buffer using 200ul wide bore filter tips, making sure to break up clumps of nuclei. Store nuclei at -80 °C for up to several months.
5m