library_construction_protocol: AT nuclei from individual participants were pooled for library preparation and sequencing to increase efficiency, cohort diversity and study power. Pooled samples were separated by condition to avoid cross-over (4-5 samples/pool; total of 6 pools/group). Sample pools for each experimental group were processed through to sequencing in lean-obese-weight loss trios to minimise between group batch effects. For each participant sample, nuclei extraction was carried out as follows. Frozen human AT (~100mg) was cut into <0.2cm pieces and homogenised with 1ml of ice-cold lysis buffer (Tris-HCL 10mM (Invitrogen, 15567-027), NaCl 10mM (Invitrogen, AM9760G), MgCl2 3mM (Invitrogen, AM9530G), 0.1% NP40 (BioBasic, NDB0385), 0.2U/µl RNase inhibitor (Roche, 03335402001)) in a glass dounce homogeniser (Merck, T2690/P0485/P1110, 15 strokes, loose then tight pestles) on ice. Following homogenisation, samples were transferred through a 100µM cell strainer (Greiner Bio-One, 542000) into a pre-chilled tube using ART wide-bore tips (Thermo Scientific, 2079G). The filtered homogenate was then transferred to 1.5ml low DNA-bind tubes (Sarstedt, 72.706.700) and centrifuged at 500g and 4C for 5mins. After lipid/supernatant removal, the nuclei pellet was resuspended in 1ml wash buffer (PBS with 0.5% BSA (Invitrogen, AM2616) and 0.2U/µl RNase inhibitor), transferred to new 1.5ml low DNA-bind tubes and re-centrifuged at 500g and 4C for 5mins. After repeat lipid/supernatant removal, the nuclei pellet was resuspended in 300µl wash buffer containing DAPI (Thermo Scientific, 62248) at 0.1µg/ml to stain nuclei, and filtered through a 35µM cell strainer into a FACS tube (Falcon, 352235) on ice. Isolated nuclei from 4-5 samples from the same experimental group were pooled prior to sorting by flow cytometry. FACS was used to clean up residual debris and lipid from isolated nuclei, and to remove doublets. Pooled nuclei were sorted on a BD FACS Aria SORP. The sheath tank was bleach cleaned prior to each run and nuclease-Free PBS (1X) (Invitrogen, AM9625) was used for sheath fluid. The 405nm laser was used to excite DAPI and emission was collected using a 450/50nm bandpass filter. Single nuclei were selected by gating on the first DAPI positive band on the DAPI vs FSC plot and then subsequently gating on SSC vs FSC and FSC A vs FSC H to ensure better debris and doublet removal. All sorts were performed using the 85-micron nozzle. The sorted nuclei were collected into a BSA and RNase inhibitor-rich collection buffer (70µl of PBS with 1.375% BSA and 2.15U/µl RNase inhibitor) in low DNA-bind tubes kept at 4C. After sorting, nuclei were centrifuged at 500g for 5mins at 4C to pellet. Supernatant was removed to leave ~40µl, which was used to resuspend pellet with a wide bore pipette tip. Pooled single nuclei suspensions were used to generate barcoded single nucleus libraries for next generation sequencing: For each pool, 5,000 to 10,000 nuclei were co-encapsulated with 10X barcoded gel beads to generate Gel beads in Emulsion (GEMs) using the 10X Chromium Controller and the 10X Genomics Single Cell 3' v3.1 kit according to manufacturer's instructions. After GEM-RT and clean-up, the quantity and fragment size distribution of amplified cDNAs derived from barcoded single-cell RNAs were assessed using the Agilent 2100 Bioanalyzer High Sensitivity DNA assay. From this cDNA, snRNAseq libraries were constructed and sequenced (Illumina NextSeq2000) in 3 batches, containing equal numbers of obese, lean and control library pools, to minimize between-group batch effects. Each unique library was sequenced to a minimum depth of >20,000 paired-end reads per nucleus (Read1 28bp + Read2 90bp, with unique dual 10bp indexes).