GSM9900693: WT iWAT biological replicate 3; Mus musculus; RNA-Seq
Liver: total RNA was isolated from frozen liver tissue using TRIzol reagent. RNA degradation and contamination were assessed by agarose gel electrophoresis, and RNA purity and integrity were evaluated using a NanoPhotometer and the RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100. iWAT: Total RNA was extracted from inguinal white adipose tissue using QIAzol Lysis Reagent according to the manufacturer's instructions. RNA quality was assessed using an Agilent 5300 Fragment Analyzer, and RNA concentration was measured using a NanoDrop spectrophotometer. One microgram of total RNA per sample was used for library preparation. Liver RNA-seq libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina. Fragmented mRNA was used as the template for first-strand cDNA synthesis with random oligonucleotide primers and M-MuLV reverse transcriptase. The RNA template was subsequently degraded using RNase H, and second-strand cDNA was synthesized using DNA polymerase I and dNTPs. Purified double-stranded cDNA was subjected to end repair, dA-tailing, and sequencing-adapter ligation. cDNA fragments of approximately 250–300 bp were selected using AMPure XP beads, PCR amplified, and purified again using AMPure XP beads to generate the final libraries. Libraries were initially quantified using a Qubit 2.0 fluorometer and diluted to 1.5 ng/µL. Library fragment-size distributions were evaluated using an Agilent 2100 Bioanalyzer, and effective library concentrations were determined by quantitative PCR. Libraries with effective concentrations greater than 3 nM and acceptable fragment-size distributions were sequenced on an Illumina NovaSeq platform using a 150-bp paired-end strategy. iWAT RNA-seq libraries were prepared using the Illumina Stranded mRNA Prep Ligation Kit. Messenger RNA was enriched from 1 microgram of total RNA by poly(A) selection using oligo(dT) magnetic beads and fragmented using fragmentation buffer. Double-stranded cDNA was synthesized using random hexamer primers, followed by end repair, phosphorylation, adapter ligation, and magnetic-bead size selection of approximately 300–400-bp fragments. Libraries were amplified by PCR for 10–15 cycles and quantified using a Qubit 4.0 fluorometer. Qualified libraries were sequenced using a 150-bp paired-end strategy on Illumina NovaSeq X Plus.