GSM5960651: 26_REC; Homo sapiens; RNA-Seq
Approximately 30 mg of adipose tissue was used for RNA extraction. Tissues were homogenized using TissueLyser (Qiagen) in 1 mL TRIzol (Sigma-Aldrich). Chloroform was added, samples were mixed, subjected to centrifugation, and the aqueous phase was transferred to a new tube. Thereafter, 1:1 70% ethanol was added, and the total volume was loaded on silica-membrane columns. RNA extraction was performed using a commercially available kit (Rneasy lipid tissue kit, Qiagen). RNA concentration and purity were assessed by absorbance at 260 and 280 nm using NanoDrop One (Thermo Fisher Scientific, Waltham, MA). Aliquots of RNA (300 ng) were analyzed using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina) as described (ref). Ribosomal RNA was removed from the sample using 35 μl of rRNA removal beads (Illumina) on a magnetic plate followed by cleanup of the ribosomal-depleted RNA with 193 μl of Agencourt RNAClean XP beads (Beckman Coulter), 70% ethanol wash, and elution into 10 μl elution buffer (Illumina). The RNA sample was fragmented for 4 min at 94°C in Elute, Prime, Fragment High Mix (Illumina) and then subjected to first-strand cDNA synthesis with 1 μl of Superscript III reverse transcriptase (Life Technologies) per sample using a thermocycler programmed to 25°C for 10 min, 50°C for 15 min, and 70°C for 15 min. Second-strand cDNA was synthesized by addition of Second-Strand Marking Master Mix, and samples were incubated at 16°C for 60 min. Samples were subjected to another bead cleanup before A-tailing and ligation of adapters as per kit instructions (Illumina). Following a third bead cleanup, samples were enriched for DNA fragments by amplification using the Illumina polymerase chain reaction (PCR) Primer Cocktail and PCR Master Mix, subjected to 98°C for 30 min, followed by a predefined cycle (98°C for 10 s, 60°C for 30 s, and 72°C for 30 s) that was repeated 3 to 15 times, on the basis of each individual sample, and finally incubated for 5 min at 72°C. Samples were cleaned and validated for DNA concentration using the Qubit dsDNA HS assay kit (Invitrogen) and for base pair size and purity using the Agilent High Sensitivity DNA chip and Bioanalyzer instrument. 38-bp paired-end sequencing on a NextSeq500