title: RNA-sequencing of subcutaneous adipose tissue from surgical biopsy near umbillicus
design: We performed RNA extraction of frozen surgical biopsy specimens at The University of North Carolina Biospecimen Processing Core. After homogenizing in TRIzol and including glycogen, we quantified RNA concentration by Qubit and quality by Agilent TapeStation. For 504 samples with an RNA integrity number (RIN) ≥6, we performed RNA-seq at The University of North Carolina High Throughput Sequencing Core. We performed polyA+ selection and library preparation (KAPA Stranded mRNA), prepared dual-barcoded individual libraries, and sequenced to an average depth of 82.6 million paired-end 150-bp reads (Illumina NovaSeq 6000 S4Xp). We used the Illumina bcd2fastq pipeline (v.2.20.0) and dropped four samples due to poor sequencing quality. We assessed RNA-seq quality by applying FastQC (v.0.11.8) and MultiQC (v.1.7). We filtered adaptor sequences and sequencing reads using Cutadapt (v.1.18), requiring a Phred quality score >20 and a minimum length of 50 bp. We used STAR (v.2.7.3a) two-pass alignment to align the reads to the hg19/GRCh37 reference, using the GENCODE v.19 annotation and ENCODE alignment parameters. We retained duplicate reads and removed unpaired alignments. Brotman et al. Diabetes (2023). PMID: 37647652