Homo sapiens
Protocols: The adipose biopsies have already be analysed in a previous study: Bruun et al. Am J Physiol Endocrinol Metab, 2006 (https://doi.org/10.1152/ajpendo.00506.200). Data and human samples were collected before (baseline) and repeated after the 15-weeks of lifestyle intervention. At least 6 hours before sampling the subjects were fasting and had not engaged in vigorous physical activity. Fasting and inactivity periods were standardized between baseline and after intervention.  Adipose tissue samples were obtained from the subcutaneous abdominal depots as described in a previous study (Bruun et al. J Clin Endocrinol Metab, 2001 (https://doi.org/10.1210/jcem.86.3.7264). Briefly the skin was anesthetized with lidocaine (10 mg/mL), before a small incision was made and a minimum of 200 mg of adipose tissue was removed under sterile conditions. Immediately after removal the adipose tissue was washed with isotonic NaCl, snap-frozen in liquid nitrogen and kept at -80 °C. Some of the material was removed and used for RNA extraction in the original study. The remaining material was stored in a temperature monitored -80 °C freezer until the start of this present study. Nuclei were isolated using a modified version of the Nuclei Isolation Kit: Nuclei PURE Prep, cat. no. NUC201 from Sigma-Aldrich. The entire protocol was carried out on ice. The tissue was lysed in ice-cold lysis buffer (Nuclei Pure Lysis Buffer, Sigma cat. no. L9286, with 1 mM DL-Dithiothreitol (DTT), Sigma cat. no. D9779, and 0.01% Triton X-100, Sigma cat. no. T1565). Homogenized for 30 seconds using an ULTRA-TURRAK TP18/10 homogenizer (20,000 rpm) for 10 minutes and filtered. Nuclei were pelleted by centrifugation and washed in wash buffer (1x DPBS, no calcium, no magnesium, Gibco cat. No. 14190, with 1% Human Serum Albumin Sigma cat. No. A1887, and 0.2 u/µL RNasin Plus RNase Inhibitor [40 u/µL], Promega cat. No. 2611). The lysate was then mixed with 1.8 M Su-crose Cushion Solution (mix of Nuclei PURE 2 M Sucrose Cushion Solution, Sigma cat. no. S9308, Nuclei PURE Sucrose Cushion Solution, Sigma cat. no. S9058 and 0.83 mM DTT). The mixture was then loaded on top of 1.8 M Sucrose Cushion Solution, and centrifuged. The supernatant was re-moved and nuclei-containing pellet was resuspended, centrifuged and filtered. The final pellet was resuspended in approximately 200 µL wash buffer and counted using NucleoCounter NC-200 (ChemoMetec cat. no. 900-0201) to determine nuclei concentration. For isolation of single-nucleus RNA, the samples were run using the 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on a 10X Chromium Controller according to manufacturer's instructions (protocol version “CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D”)
ENA first public
2026-02-12
INSDC center name
Novo Nordisk A/S
INSDC status
public
Submitter Id
E-MTAB-16620:A_Before
broker name
ArrayExpress
clinical history
Before Lifestyle intervention
collection date
not collected
common name
human
developmental stage
adult
geographic location (country and/or sea)
not collected
individual
A
isolate
not applicable
organism part
subcutaneous adipose tissue
sample name
E-MTAB-16620:A_Before
scientific_name
Homo sapiens
sex
female