Computational protocol: SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes

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Protocol publication

[…] Total RNA was extracted from SGBS adipocytes (in duplicates) and PHWSC adipocytes (n = 4 derived from 3 females and 1 male with an average age of 39.5 ± 15.9 years old) using Qiagen RNeasy plus kit (Qiagen Inc, CA, USA). Poly-A mRNA was then enriched with oligodT beads (Life Technologies) from approximately 5 µg of total RNA. 100 ng of poly-A mRNA recovered was used to construct multiplexed strand-specific RNA-Seq libraries as per manufacturer’s instructions (NEXTflexTM Rapid Directional RNA-Seq Kit (dUTP-Based) v2). Individual library quality was assessed with an Agilent 2100 Bioanalyzer and quantified with a QuBit 2.0 fluorometer before pooling for sequencing on a HiSeq 2000 (1 × 101 bp read). The pooled libraries were quantified using the KAPA quantification kit (KAPA Biosystems) prior to cluster formation. 20–26 million reads were mapped in all samples with a rate of >95%. Fastq-formatted reads were processed with Trimmomatic to remove adapter sequences and trim low quality bases (LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 4:15 MINLEN: 36). Reads were aligned to the human genome (hg19) using Tophat version 2 (settings–no-coverage-search–library-type = fr-firststrand). Feature read counts were generated using htseq-count (Python package HTSeq default union-counting mode, strand = reverse). Differential Expression analysis was performed using the edgeR package in both ‘classic’ and generalized linear model (glm) modes to contrast the SGBS cell strain and primary human subcutaneous adipocytes (PHWSC adipocytes) derived from obese subjects. Genes with at least 1 count per million reads were included for edgeR analysis. Unsupervised clustering was performed using the heatmap.3 function from the GMD R package. The raw htseq-counts were first normalized using variance stabilizing transformation from the DESeq2 package before cluster analysis. iPathwayGuide (Advaita bioinformatics) and DAVID Bioinformatics, web-based analysis tools were further employed to identify the top biological pathways and metabolic pathways respectively. KEGG pathway interaction map of metabolism related genes was built in Cytoscape version 3.3 using CluePedia and ClueGo plugin, . [...] Total RNA was isolated using Qiagen RNeasy plus kit and quantified using nano-drop 2000 spectrophotometer (Thermo Scientific, IL, USA). cDNA was synthesized from 800 ng of RNA using ABI high capacity cDNA synthesis kit (Applied Biosystems, CA, USA). Real-time quantitative polymerase chain reaction (qPCR) using Qiagen QuantiFast SYBR Green PCR Kit was employed to quantify relative gene expression in samples. Primers involved in various pathways of interest such as adipogenesis (PPARγ, CEBPα, Adiponectin, Leptin), lipid storage (FABP4, FITM2) and browning (UCP1, PGC1α, DIO2) were prioritized in this study, , , , , –. NCBI primer blast and ABI primer express 3.0 were used to generate primer sequences (Supplementary Table ).Differences in mitochondrial DNA content between SGBS and PHWSC adipocytes was determined by real-time PCR using TaqMan probes specific for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA or 18 s rRNA) as previously described (Supplementary Table ). Taqman probes were labelled at the 5′ end with fluorescent reporter FAM and the 3′ ends were labelled with a quencher TAMRA. mtDNA content was measured by the ratio of relative expressions of mtDNA to that of nDNA. […]

Pipeline specifications

Software tools Trimmomatic, TopHat, HTSeq, edgeR, DESeq2, iPathwayGuide, CluePedia, ClueGO, Primer-BLAST, Primer Express
Databases KEGG PATHWAY
Applications RNA-seq analysis, qPCR
Organisms Homo sapiens
Chemicals Glucose, Isoproterenol, Oxygen