title: GSM8705122: Adult_ob, HET, rep3, RNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: The P0 pups or adult mice were decapitated. The skin and skull of the head were carefully removed, and the brain was placed in the 35-mm dish filled with ice-cold PBS. The cortical or olfactory bulb tissues were then dissected out using fine forceps and immediately transferred to a new microcentrifuge tube containing ice-cold TRIzol Reagent. The sample was homogenized using a pestle and then centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was transferred into a new microcentrifuge tube, and 100 l of chloroform was added. The sample was then incubated at room temperature for 3 min after vigorous shaking for 15 sec. After centrifuging at 12,000 × g for 15 min at 4°C, the upper aqueous phase was carefully transferred into a new RNase-free microcentrifuge tube, followed by the addition of 250 l of isopropanol. The mixture was inverted several times and incubated at room temperature for 10 min. After centrifuging at 12,000 × g for 10 min at 4°C, the supernatant was discarded, and the RNA pellet was washed once with 1 ml of freshly-made 75% ethanol, air-dried for 5 min, and dissolved in 30 l of nuclease-free water. The concentration of RNA was measured using the spectrophotometer. The high-quality RNA, with an A260/A280 ratio of ~2.0, is subjected to library preparation. The polyadenylated mRNA was isolated using oligo(dT) coupled to magnetic beads from 1 g of total RNA and fragmented by heating at 94°C for 8 min. The first cDNA strand was synthesized by reverse transcription with a random primer (N6). After synthesizing the second strand DNA, the Y-shaped adapter-HTS was ligated to both ends. The ligated DNA was cleaned by AMPure XP Beads. Finally, the RNA-seq library was synthesized through PCR (98°C, 30 s; 98°C, 10 s, 60°C, 30 s, 72°C, 30 s for 10 cycles; and a final extension at 72°C, 5 min) by combining the reagents (2.5 l of P5 primer, 2.5 l of P7 primer, and 25 l of Amplification Mix). All RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 platform.