Isolation of human DRG nuclei

Isolation of neuronal nuclei from human DRGs (or tough to dissociate tissues):

Freeze tissue on dry ice and store at -80 ^oC until use. 

Alternatively, cut tissues into small pieces (1-2 mm) and incubate in RNAlater (ThermoFisher, cat# AM7021) overnight at RT 

Remove excess RNAlater and freeze tissue pieces in a microfuge tube on dry ice. 

Store at -80 ^oC until use 

On day of 10x run: prepare homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 10 mM Tris, pH 8.0, 1 µM DTT, 0.1% Triton X-100 (v/v). 

Cool on ice. Stock solutions can be prepared ahead of time. 

Cool down all necessary equipment and solutions. 

Cool Spectrum™ Bessman Tissue Pulverizer (ThermoFisher, cat#08-418-3) in liquid nitrogen or dry ice 

Add frozen tissues to Spectrum™ Bessman Tissue Pulverizer (ThermoFisher, cat#08-418-3) in liquid nitrogen or dry ice Pulverize tissues and transfer to glass dounce homogenizer (Fisher Scientific, cat#357538) containing 1ml of cold homogenization buffer 

Homogenize tissues in glass dounce homogenizer in 1ml of cold homogenization buffer: 5 strokes with “loose” pestle and ~10-20 strokes with “tight” pestle … on ice 

Filter through 40 µm cell strainer (ThermoFisher Scientific, cat# 08-771-1) 

Transfer to microfuge tube (low bind; Sorenson BioScience, cat# 11700) and spin @ ~300-800g at 4 o for 5-8 mins (determine what speed and time are best for your tissues) 

Remove supernatant and resuspend pellet in 500 µl of PBS + 1% BSA + SUPERaseIn RNase Inhibitor ( 0.2 U/µ; ThermoFisher Scientific, Cat# AM2696) 

Incubate on ice for 10-15 mins 

Add rabbit polyclonal anti-NeuN antibody (Millipore, cat#ABN78) 1:4000 to 1:5000 Incubate with rotation at 4 ^oC for 30 mins Spin @ 300-800g at4 ^oC  for 5-8 mins 

Remove supernatant and “wash” by adding 1 ml PBS + 1% BSA + SUPERaseIN RNase inhibitor 

Spin @ ~300-800g at 4 ^oC  for 5-8 mins 

Resuspend pellet in 80 µl of PBS + 0.5% BSA + 2mM EDTA. 

Volume is for 10⁷ total cells. 

Adjust volumes accordingly if you have more total cells. 

Add 20 µl of anti-rabbit IgG Microbeads (Miltenyi Biotech, cat# 130-048-602). 

Volume is for 10⁷ total cells. 

Adjust volumes accordingly if you have more total cells. 

Incubate at 4 ^oC 15-20 mins 

“Wash” by adding 1 ml of PBS + 0.5% BSA + 2mM EDTA Spin @ ~300-800g at 4 ^oC for 5-8 mins 

Remove supernatant and resuspend in 0.5-1 ml PBS + 0.5% BSA + 2mM EDTA 

Load onto LS column (Miltenyi Biotec, cat# 130-042-401) – follow instruction from manufacturer: put column on magnetic MidiMACS separator (Miltenyi Biotec, cat#130-042-302; MACS MultiStand (cat#130-042-303), add 3 ml of ice cold buffer (PBS + 0.5% BSA + 2mM EDTA) to equilibrate 

Add nuclei to column 

Wash column with 3 ml ice cold buffer (3x) 

Remove column from magnet and elute in 5 ml of same buffer using plunger that comes with the LS column. 

Elute on ice into 15 ml Falcon tube. 

Spin @ 500g at  4 ^oC for 10 mins 

Remove supernatant 

Resuspend in 1.5 ml of PBS + 1% BSA + SUPERaseIn RNase Inhibitor. 

Spin @ 300-800g at  4 ^oC for 5-8 mins 

Resuspend in 1-1.5 ml of PBS + 1% BSA + SUPERaseIn 

RNase Inhibitor Strain through 35 µm cell strainer (Falcon 352235) to remove any nuclei clumps. 

Strain through 35 µm cell strainer (Falcon 352235) to remove any nuclei clumps. 

Alternatively, use Ultra-Turrax (Laboratory Supply Network, Inc., cat#IKA:3737001) on setting 1 for 10-45 sec on ice (adjust time accordingly depending on how many nuclei you have) 

1st count: Stain 5-10 µl with trypan blue and count (using a hemocytometer) also check for clumps 

Transfer to low bind microfuge tube and spin @ ~300-800g at 4 ^oC for 5-8 mins 

Remove supernatant and resuspend in desire volume with PBS + 1% BSA + SUPERaseIn RNase Inhibitor (volume based on the first count.) 

Calculate for 2000 nuclei/µl; the first count is not that accurate (too diluted), so this allows final dilution to 1000 nuclei/µl. Stain 5-10µl with trypan blue and do a 2nd count. 

Adjust volume to desire nuclei concentration (e.g. 1000 nuclei/µl).

Optional: do a 3rd count. 

This should be close to the 2nd count. 

Load appropriate number of nuclei onto 10X chip. 

Homogenization Buffer: 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris, pH 8.0, 1 µM DTT, 0.1% Triton X-100

Can also use EZ PREP buffer (Sigma, Cat #NUC-101) as the homogenization buffer. 

This is a modification of the protocol described in Nguyen et al (2019) Stereotyped transcriptomic transformation of somatosensory neurons in response to injury eLife 8:e49679