title: GSM8416022: HFrST2, High fructose diet, Stress; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using a mirVanaTM isolation kit as per protocol (Catalog number: #AM1560; Thermo Fisher Scientific). To prepare the RNA samples, we added 10x DNase I buffer and one unit of DNase I enzyme (New England Biolabs) to a maximum of 5 μg RNA for DNase treatment. The samples were incubated at 37°C for 15 minutes and then the enzyme was inactivated at 70°C for 15 minutes. After that, we quantified the DNase-treated RNA by measuring the absorption at 260 nm using a NanoDrop 2000 spectrophotometer. The MGIEasy RNA Library Prep Set (MGI) was usedfor library preparation following the manufacturer's instructions. Initially, 500 ng of total RNAwas used, and the rRNAwas depletedusing an MGIEasy rRNAdepletion kit. Following rRNA depletion, samples were fragmented and reverse transcribed. The second strand was then synthesized and converted into cDNA. The kit provided DNA Clean Beads for DNA purification, followed by end repair and A-tailing. The samples were then barcoded, adaptor-ligated, and subjected to purification. Adaptor-specific primers were used for amplification, and quantification was done using a Qubit dsDNA high-sensitivity kit from (Thermo Scientific). The size of the sample fragments was determined using a 4200 TapeStation (Agilent). Denaturation and circularization of 1pMol dsDNAwere performedto generate single-stranded circular DNA, followed by the creation of DNA Nano Balls using Rolling cycleamplification. The DNBs were sequenced on an MGISEQ-2000 sequencer (MGI) using the PE100 recipe, after being placed on a patterned flow cell.