Single nuclei isolation from frozen human adipose tissue for 10x Genomics multiome sequencing

Prepare all the buffers listed in the tables below. Any buffers that contain RNase Inhibitor must be prepared the day of the nuclei isolation. Buffers without RNase Inhibitor may be prepared a day ahead.

NP40 Lysis Buffer: (A)

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 10mL
Tris-HCl (pH 7.4)	Sigma	T2194-100ML	1 M	10 mM	100uL
NaCl	Promega	V4221	5 M	10 mM	20uL
MgCl2	Sigma	M1028-100mL	1 M	3mM	30uL
Nonidet P40 Substitute	G-Biosciences	DG001	10%	0.1%	100uL
DTT	Sigma	43816-10mL	1000 mM	1 mM	10uL
Nuclease-free water	Invitrogen	10977			9490uL
RNase inhibitor	Sigma	3335402001	40 U/uL	1U/uL	250uL

DPBS/1% BSA/1U/uL RNase Inhibitor Buffer:

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 3mL
DPBS-/-	Gibco	14190-144	1X		2625uL
BSA*	Sigma	A7030-100G	10%	1%	300uL
RNase inhibitor	Sigma	3335402001	40 U/uL	1U/uL	75uL

• Bovine Serum Albumin powder dissolved in DPBS-/- and filter sterilized.

Lysis Buffer (to prepare 0.1 X Lysis Buffer): (B)

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 0.5mL
Tris-HCl (pH 7.4)	Sigma	T2194-100ML	1 M	10 mM	5uL
NaCl	Promega	V4221	5 M	10 mM	1uL
MgCl2	Sigma	M1028-100mL	1 M	3mM	1.5uL
Tween-20	Bio-Rad	1610781	10%	0.1%	5uL
Nonidet P40 Substitute	G-Biosciences	DG001	10%	0.1%	5uL
Digitonin	Invitrogen	BN20061	5%	0.01%	1uL
BSA	Sigma	A7030-100G	10%	1%	50uL
DTT	Sigma	43816-10mL	1000 mM	1 mM	0.5uL
Nuclease-free water	Invitrogen	10977			418.5uL
RNase inhibitor	Sigma	3335402001	40 U/uL	1U/uL	12.5uL

Lysis Dilution Buffer (to prepare 0.1 X Lysis Buffer): (C)

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 1mL
Tris-HCl (pH 7.4)	Sigma	T2194-100ML	1 M	10 mM	10uL
NaCl	Promega	V4221	5 M	10 mM	2uL
MgCl2	Sigma	M1028-100mL	1 M	3mM	3uL
BSA	Sigma	A7030-100G	10%	1%	100uL
DTT	Sigma	43816-10mL	1000 mM	1 mM	1uL
Nuclease-free water	Invitrogen	10977			859uL
RNase inhibitor	Sigma	3335402001	40 U/uL	1U/uL	25uL

0.1 X Lysis Buffer:

A	B	C	D	E
Ingredient	Source	Stock Conc.	Final Conc.	Volume for 1mL
1 X Lysis Buffer (B)	Prepared in lab	1 X	0.1 X	100uL
Lysis Dilution Buffer (C)	Prepared in lab			900uL

Wash Buffer: (D)

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 2mL
Tris-HCl (pH 7.4)	Sigma	T2194-100ML	1 M	10 mM	20uL
NaCl	Promega	V4221	5 M	10 mM	4uL
MgCl2	Sigma	M1028-100mL	1 M	3mM	6uL
BSA	Sigma	A7030-100G	10%	1%	200uL
Tween-20	Bio-Rad	1610781	10%	0.1%	20uL
DTT	Sigma	43816-10mL	1000 mM	1 mM	2uL
Nuclease-free water	Invitrogen	10977			1698uL
RNase inhibitor	Sigma	3335402001	40 U/uL	1U/uL	50uL

2X Diluted Nuclei Isolation Buffer:

A	B	C	D	E	F
Ingredient	Source	Ref. No.	Stock Conc.	Final Conc.	Volume for 0.2mL
Nuclei Buffer*	10X Genomics	2000153/ 2000207	20x	2x	20uL
DTT	Sigma	43816-10mL	1000 mM	2 mM	0.4uL**
RNase inhibitor	Sigma	3335402001	40 U/uL	2 U/uL	10uL
Nuclease-free water	Invitrogen	10977			169.6uL

• Nuclei Buffer supplied in the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent bundle.** Can dilute DTT 1:5 in water and use 2uL. Reduce water volume to 168uL.

Transfer frozen adipose tissue to a 300mL porcelain mortar pre-chilled and filled with liquid nitrogen. Press/tap on the frozen tissue with a pre-chilled porcelain pestle to pulverize it. A little grinding may be necessary.

Transfer the pulverized tissue to an ice-cold 15mL conical glass grinder tube, (using a pre-chilled metal spatula), just after the liquid nitrogen disappears.

Immediately add 2mL of cold NP40 Lysis Buffer to the tissue.

Homogenize the tissue/cells in the grinder tube with a cold conical PTFE pestle. Use twisting strokes with the pestle. The number will be determined by the appearance of tissue, (e.g.,12 strokes).

Tips:

• keep on ice all the time, (the conical grinder tube should be immersed in ice)

• use gentle strokes, (with pressure if necessary)

• avoid foaming

• add more NP40 Lysis Buffer, if necessary, (not more than 900uL)

• use the number of strokes necessary to reach the bottom of the tube with the pestle

Using a 2mL pipet with a wide bore, transfer the liquid portion of the homogenate only to a 100um cell strainer (Biologix, Ref. No. 15-1100, or other) pre-wetted with 150uL of NP40 Lysis Buffer and placed on a 50mL conical polypropylene centrifuge tube.

Wash the contents of the conical glass grinder tube with 500uL of NP40 Lysis Buffer and transfer this to the strainer as well.

Wash the strainer with 500uL of NP40 Lysis Buffer.

Pass the filtered homogenate through a 40um cell strainer (Falcon, Ref. No. 352340, or other) pre-wetted with 150uL of NP40 Lysis Buffer into another 50mL conical polypropylene centrifuge tube. Wash the strainer with 500uL of NP40 Lysis Buffer.

Transfer the contents of the 50mL tube to a 15mL polypropylene centrifuge tube.

Centrifuge at 500 rcf for 5 min at 4℃ in a swinging-bucket rotor.

Carefully remove any lipid layer with a plastic transfer pipet, then carefully remove the supernatent down to about 50uL using a 1mL pipet tip.

Add 1mL of DPBS/1% BSA/1U/uL RNase Inhibitor, but DO NOT mix.

Incubate on ice for 5 min.

Pipette mix to resuspend pellet (5x). Leave the sample in the 15mL centrifuge tube.

Centrifuge at 500 rcf for 5 min at 4℃.

Remove the supernatent.

Resuspend in 400uL of DPBS/1% BSA/1U/uL RNase Inhibitor and pipet to mix (5x). Keep on ice.

Repeat steps 2-18 for additional samples.

Transfer 25uL of unstained nuclei sample to a 5mL polystyrene, round-bottom (FACS) tube (Corning Falcon, Ref. No. 352008) containing 250uL of DPBS/1% BSA to reserve unstained. This only needs to be performed for one sample.

Transfer the rest of the nuclei prep, plus 100uL of DPBS/1% BSA/1U/uL RNase Inhibitor used to rinse the 15mL tube, to another 5mL FACS tube. Add 7AAD Ready-Made Solution (Sigma-Aldrich, Ref. No. SML1633) to this nuclei sample, (1uL 7AAD per 100uL sample). 7-AAD is excited with a 488 laser and emission is detected around 647nm.

Incubate for at least 5 minutes on ice with light exclusion.

Fill a 5mL FACS collection tube with DPBS/5% BSA and incubate for 5 minutes to coat the collection tubes with protein.

Remove the DPBS/5% BSA from the collection tube and rinse with PBS/1% BSA.

Pipet enough DPBS/10% BSA and 40U/uL RNase Inhibitor into the collection tube to achieve a final concentration of 1% BSA and 1U/uL RNase Inhibitor after the addition of the sorted nuclei. Volumes should be determined empirically through testing of FACS.

Proceed to FACS machine for nuclei sorting. Use a 100um nozzle. See the gating strategy below.

Transfer sorted nuclei to a 15mL polypropylene tube and centrifuge at 500 rcf for 5 min at 4℃.

Remove the supernatent without disrupting the nuclei pellet down to about 25uL. The pellet will not be visible.

Resuspend the pellet in 100uL of 0.1X Lysis Buffer and pipet to mix (5x).

Incubate for 2 min on ice.

Add 1mL of Wash Buffer and pipette to mix (5x).

Centrifuge at 500 rcf for 5 min at 4℃.

Remove the supernatent without disrupting the nuclei pellet down to about 25uL.

Resuspend the pellet in an equal volume of 2X Diluted Nuclei Isolation Buffer. Pipet to mix (5x). The nuclei are now ready for counting and proceeding to the CG000338 Chromium Next GEM Multiome ATAC + GEX User Guide (Version F).

After sorting and permeabilizing the nuclei, they should appear to be mostly single and intact. We obtained about 1.4 x 10⁵ nuclei/gram of omental adipose tissue, and 7.4 x 10⁴ nuclei/gram of subcutaneous abdominal adipose tissue.