Transcriptomic profiling of brown adipose tissue in high-fat diet-induced obese mice following Pu-erh tea extract intervention
Background: Obesity driven by high-fat diet (HFD) is a global public health concern, and Pu-erh tea extract (PTE) exhibits anti-obesity potential, but its regulatory mechanism on brown adipose tissue (BAT) metabolism remains unclear. Transcriptomic analysis of BAT is critical to reveal PTE-mediated metabolic remodeling. Methods: C57BL/6J mice were divided into HFD (high-fat diet) and HFD+PTE groups. BAT samples were collected after 16-week intervention for RNA sequencing. Results: Transcriptomic data showed 623 differentially expressed genes (DEGs) in HFD+PTE vs HFD groups, mainly enriched in cAMP/Calcium signaling, Biosynthesis of unsaturated fatty acids, and Glycerolipid metabolism pathways. Value: This dataset provides transcriptomic evidence for PTE regulating BAT function, complementing our previously deposited gut microbiota 16S rRNA data, and facilitating research on natural product-mediated metabolic regulation. Overall design: 1. Study Overview This study aimed to elucidate the transcriptomic alterations in brown adipose tissue (BAT) of high-fat diet (HFD)-induced obese mice following intervention with Pu-erh tea extract (PTE), and to explore the molecular mechanisms underlying PTE-regulated metabolic remodeling of BAT. The transcriptomic data submitted here correspond to BAT samples isolated from 8 mice (4 mice per group), capturing the differential gene expression profiles associated with the anti-obesity effects of PTE.  2. Sample Preparation  Animal Model: Specific pathogen-free (SPF) male C57BL/6J mice (6 weeks old, 17±2 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were acclimatized for one week under standard conditions (22±2°C, 12-h light/dark cycle) with free access to food and water.  Grouping and Intervention: Mice were randomly assigned to two groups (n=4 per group):  HFD Group: Fed a high-fat diet (60% kcal from fat) for 16 weeks.  HFD+PTE Group: Fed the high-fat diet for 16 weeks with ad libitum access to drinking water supplemented with 0.25% (w/v) PTE. The PTE was prepared from ripened Pu-erh tea (7572, Yunnan TEATEA Group, production year 2006) via sequential hot-water extraction (1:15 and 1:10, w/v), followed by filtration, combination of filtrates, and lyophilization. The resulting extract was stored at -80°C until use.  3. Tissue Collection Following the 16-week intervention and a 12-hour fast, mice were euthanized by CO2 inhalation. Interscapular brown adipose tissue (BAT) was rapidly dissected, rinsed briefly with ice-cold sterile saline, blotted dry, and immediately snap-frozen in liquid nitrogen. All samples were stored at -80°C until RNA extraction.  4. RNA Extraction and Quality Control Total RNA was extracted from frozen BAT samples using TRIzol® Reagent according to the manufacturer's instructions. RNA concentration and purity were measured using an ND-2000 spectrophotometer (NanoDrop Technologies). RNA integrity was assessed with a 5300 Bioanalyzer (Agilent Technologies). Only RNA samples meeting stringent quality criteria (OD260/280 ratio of 1.8-2.2, OD260/230 ratio = 2.0, RNA Quality Number (RQN) = 6.5, 28S:18S ribosomal RNA ratio = 1.0, and total mass > 1 µg) were proceeded to library construction.  5. Library Construction, Sequencing, and Data Processing Sequencing libraries were prepared from 1 µg of qualified total RNA using the Illumina® Stranded mRNA Prep, Ligation kit, following the manufacturer's protocol. This included polyA selection, RNA fragmentation, double-stranded cDNA synthesis, adapter ligation, size selection (~300 bp), and PCR amplification. Library quantification was performed using Qubit 4.0. Pooled libraries were sequenced on an Illumina NovaSeq X Plus platform using a NovaSeq Reagent Kit to generate 150-bp paired-end reads. Raw reads were processed with fastp for quality control and adapter trimming. Clean reads were aligned to the GRCm39 reference genome using HISAT2. Transcript assembly and quantification (in TPM) were performed using StringTie and RSEM, respectively. Differential expression analysis was conducted with DESeq2, with significance thresholds set at |log2FC| = 1 and FDR < 0.05. Alternative splicing events were analyzed using rMATS. Functional enrichment analysis (GO and KEGG) was performed on differentially expressed genes.