1. Animal Preparation: C57BL/6J mice were acclimated for two weeks at room temperature (22°C) and then randomly assigned to cold (6.5°C) or CL-316,243 (CL, 1 mg/kg) treatment.
2. Tn5 Transposase Preparation: Tn5 transposase was produced and loaded with MEA and ME-B oligonucleotides.
3. Nuclei Isolation: Inguinal adipose tissues were collected, minced, and homogenized to extract nuclei.
4. snATAC-seq: The snATAC-seq was performed with combinatorial indexing, where nuclei were tagged, sorted, and amplified.
5. Sequencing: Libraries were sequenced using a NextSeq 500 sequencer (Illumina).
6. Data Preprocessing: Sequencing data was demultiplexed, aligned to the mm10
reference genome, and quality checked.
7. Quality Control: Cells were filtered based on read depth and doublet scores to
remove low-quality cells and potential doublets.
8. Clustering and Annotation: Dimensionality reduction and clustering were performed to identify cell types based on chromatin accessibility profiles.
9. Differential Analysis: Differentially accessible genes and peaks were identified between cell types and conditions.
10. Transcription Factor Analysis: Transcription factor motif enrichment and activity were analyzed using chromVAR.
11. Co-accessibility Analysis: Co-accessibility of peaks was assessed using Cicero to identify potential regulatory interactions.
12. Trajectory Analysis: Pseudotime trajectory analysis was performed to infer the developmental trajectory of beige adipocytes.
13. Lipidomics: Lipids were extracted from adipose tissue and analyzed using gas chromatography-mass spectrometry (GC-MS).
1. Animal Preparation: C57BL/6J mice were acclimated for two weeks at room temperature (22°C) and then randomly assigned to cold (6.5°C)
or CL-316,243(CL, 1 mg/kg) treatment.
2. Tn5 Transposase Preparation: Tn5 transposase was produced and loaded with MEA and ME-B oligonucleotides.
3. Nuclei Isolation: Inguinal adipose tissues were collected, minced, and homogenized to extract nuclei.
4. snATAC-seq: The snATAC-seq was performed with combinatorial indexing, where nuclei were tagged, sorted, and amplified.
5. Sequencing: Libraries were sequenced using a NextSeq 500 sequencer (Illumina).
6. Data Preprocessing: Sequencing data was demultiplexed, aligned to the mm10 reference genome, and quality checked.
7. Quality Control: Cells were filtered based on read depth and doublet scores to remove low-quality cells and potential doublets.
8. Clustering and Annotation: Dimensionality reduction and clustering were performed to identify cell types based on
chromatin accessibility profiles.
9. Differential Analysis: Differentially accessible genes and peaks were identified between cell types and conditions.
10. Transcription Factor Analysis: Transcription factor motif enrichment and activity were analyzed using chromVAR.
11. Co-accessibility Analysis: Co-accessibility of peaks was assessed using Cicero to identify potential regulatory interactions.
12. Trajectory Analysis: Pseudotime trajectory analysis was performed to infer the developmental trajectory of beige adipocytes.
13. Lipidomics: Lipids were extracted from adipose tissue and analyzed using gas chromatography-mass spectrometry (GC-MS).91