title: I_Before
organism: Homo sapiens
description: 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”)
attributes.tag: ENA first public
attributes.value: 2026-02-12
attributes.tag: INSDC center name
attributes.value: Novo Nordisk A/S
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-16620:I_Before
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: clinical history
attributes.value: Before Lifestyle intervention
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: I
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: subcutaneous adipose tissue
attributes.tag: sample name
attributes.value: E-MTAB-16620:I_Before
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male