Agilent s 2100 bioanalyzer

Total RNA samples were extracted from the nine harvested tissues using Qiagen’s RNeasy Mini Kit. Due to the presence of genomic DNA in the RNA extract, a modified protocol was employed by utilising Epicentre’s DNase I solution to remove genomic DNA contamination. The intactness of the RNA extracts were visualised using 1% agarose gel, and their respective concentrations quantitated using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). The integrity of the RNA extracts were determined using Agilent’s 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) via an RNA Pico chip. RNA samples with RIN of more than 7.0 were selected for the subsequent library preparation step.

Mice were euthanized at the end of 4-week feeding. Three of five mice from each dietary group with greatest, medium, and least fat mass were used for RNA-Seq in the current study. In order to be consistent, right side of BAT at the interscapular region was collected, weighed, flash frozen, and stored at −80 °C. Total RNA was isolated for sequencing using E.Z.N.A^® Total RNA kit II (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. Briefly, ~20 mg BAT were homogenized in 1 mL of RNA-solv reagent using bullet blender^® tissue homogenizer (Next Advance Inc., Averill Park, NY, USA). The homogenized tissues were then processed with chloroform addition and phase separation. The aqueous phase was transferred to a new sterile 1.5 mL tube and mixed with equal volume of 70% ethanol. The mixtures were loaded to the Hibind^® RNA spin column and processed according to the manufacturer’s instruction. Total RNA was eluted in 30 μL nuclease-free water and stored in −80 °C. The concentration and purity of extracted RNA samples were checked using a NanoDrop^TM spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), then analyzed using an Agilent RNA 6000 pico kit with an Agilent’s 2100 Bioanalyzer (Agilent Technologies Inc., Waldbronn, Germany). High quality of RNA samples with an RNA integrity number (RIN) above 6.5 were used for analysis.

For library generation, Illumina TruSeq DNA PCR-Free HT Library Prep Kit (Illumina, San Diego, CA) was used according to the manufacturer’s standard protocol and obtained high-quality libraries using 500 ng of starting material (during optimization, input amounts as low as 100 ng were tested and showed no loss of quality on the QC level). Qubit 1.0 (Thermo Fisher Scientific Inc., Schwerte, Germany) was used for quantification, Agilent’s 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) for quality assessment and Kapa HIFI Library quantification kit (Kapa Biosystems Inc., Wilmington, MA) for final quantification before pooling. Libraries were pooled equimolarly. Sequencing of the libraries was performed on an Illumina MiSeq using Reagent Kit v3 (Illumina, San Diego, CA; 600 cycles) in paired-end mode, with 30% PhiX added.

A total of nine sequencing libraries were prepared, with three replicates for each tissue type. The library preparation step consisted of two main sections; the removal of ribosomal RNA (rRNA) from the total RNA, and the conversion of the rRNA-depleted RNA to cDNA. rRNA depletion was carried out using the Ribo-Zero™ Gold Kit (Human/Mouse/Rat) (Epicentre, Madison, WI, USA), while the conversion of treated RNA to cDNA was performed using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI, USA). The size fragmentation distribution of the constructed cDNA library was assessed using Agilent’s 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) via a High-Sensitivity DNA assay. The cDNA library was also subjected to quantitation via a RT-qPCR assay using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The final normalised cDNA library was pooled together in a single reaction and was sequenced on Illumina’s HiSeq 2500 platform in rapid run mode for 75 × 2 bp paired-end reads at the High Impact Research Centre in Universiti Malaya, Malaysia. Paired end sequencing data for lymph node, spleen, and thymus were submitted to NCBI Short Read Archive (SRA) database under accession SRP096937.

AGS cells were treated with DAA for 48 h, and total RNA was purified using RNAiso Plus reagent (Takara, Shiga, Japan) according to the manufacturer’s instructions. RNA-Seq was commercially commissioned by Ebiogen (EBIOGEN, Seoul, Korea). Total RNA quality was measured using an Agilent’s 2100 bioanalyzer. Library construction was performed using the Quant-Seq library prep kit (Lexogen, Vienna, Austria). Next, we evaluated high-accuracy sequencing with high-throughput single-end 75 bp sequencing using NextSeq 500 (Illumina, San Diego, CA, USA). After sequencing, the differences in gene expression were analyzed using Exdega (EBIOGEN, Seoul, South Korea). For the identification of DEGs, data were normalized and quantified using the LPEseq package [26 (link)]. Gene classification was based on searches performed by DAVID (david.abcc.ncifcrf.gov (accessed on 16 December 2020)) and Medline databases (ncbi.nlm.nih.gov (accessed on 12 January 2021)).