Adipocyte
nuclei isolation from iWAT- 200-400
mg of inguinal white adipose tissues (iWAT) from mice exposed
to conditions mentioned in the text were placed
on sterile 6-well tissue culture plate with ice-cold 1XPBS. Fat pads
were blotted on a napkin to removed excess liquid. Tissues were cutand mincedwith scissors andwere placed in 15ml conical tubes
containing digestion buffer (DPBS and
Collagenase D at 9.8 mg/ml; Sigma, MO)at incubated
at 37 C for 45 mins with gentle
shaking at 100 rpm.
10 ml of resuspension media (DMEM/F12
with glutamax supplemented with 15% FBS and 1% pen/strep;
Thermo Scientific, CA) was added to
digested solution and slowly inverted 5 times. The digestion mixture
was centrifuged at 200 x g for 5mins at RT. Floating adipocytes were
collected using P1000 pipet with half cut P1000 tip. Adipocytes were
transferred to a new 15 ml tube and kept on ice for 5 mins. Excess
liquid was aspirated using 1 ml syringe and adipocytes were then
washed with 1ml DPBS and the suspension was spun down at 200 g for
mins at RT. Spun down liquid was aspirated using 1 ml syringe and
adipocyte nuclei were isolated using Minute nuclei and cytosol
isolation kit for adipose tissue using manufacture’s instruction
(Invent Biotechnologies, MN) with modifications. Briefly, adipocytes
were slowly resuspended in 600 l
nuclei lysis buffer (N/C Buffer) and lysate was transferred to a
filter cartridge with collection tube and incubated at -20°C freezer
for 20 min with cap open. After incubation, the tube was centrifuged
at 2000 rpm for 2 min at 4 °C. The filter cartridge was discarded
without agitation and the collection tube was immediately centrifuged
at 4000 rpm for 4 min at 4 °C. Supernatant was gently removed using
P200 pipet without touching the side walls. Nuclei were resuspended
in 30 ul of nuclei resuspension buffer (DPBS+0.1%BSA) per 200-400 mg
of iWAT (i.e. one 8-10 week chow fed mouse). For SNAP-seq, 2-3 mice
were combined and 60 l of nuclei
suspension was transferred to a new 2 ml tube and resuspended with
500-700 l of nuclei resuspension
buffer and filtered using 40 m cell
strainer (Flowmi Cell Strainer, Belart, NJ) twice to get clean single
nuclei suspension. As shown in Figure 3A, for quality control,
nuclei were first DAPI stained and then filtered or FACS sorted to
get single nuclei suspension. After microfluidic partitioning in
10xGenomics platform (see below), nuclei lysis was checked by
observing oil emulsion under fluorescent microscope for DAPI
diffusion.
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How to cite:
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
Rajbhandari, P(2020). Adipocyte nuclei isolation from iWAT. Bio-protocol Preprint. bio-protocol.org/prep225.
Rajbhandari, P., Arneson, D., Hart, S. K., Ahn, I. S., Diamante, G., Santos, L. C., Zaghari, N., Feng, A., Thomas, B. J., Vergnes, L., Lee, S. D., Rajbhandari, A. K., Reue, K., Smale, S. T., Yang, X. and Tontonoz, P.(2019). Single cell analysis reveals immune cell–adipocyte crosstalk regulating the transcription of thermogenic adipocytes. eLife. DOI: 10.7554/eLife.49501
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