GSM9017179: Liver, Hoip_fl/fl.AdipoQ.Cre_positive, 16 weeks HFD 60%, biol rep 5; Mus musculus; RNA-Seq
At 16 weeks of diet, livers were collected, snap frozen in liquid nitrogen, and kept at -80 degrees until RNA extraction. The liver samples were processed according to the manufacturer's instructions using the RNeasy Midi kit (Qiagen, 75142). For tissue disruption 100 mg of liver were resuspended in 2 ml of lysis buffer RLT plus with β-mercaptoethanol (10 ul β-ME were added to every 1 ml RLT buffer) samples were homogenised in TissueLyser II using a 5 mm bead in two rounds for 2 minutes at 30 1/s. For both tissues a DNase digest kit was employed, involving the addition of 350 µl of Buffer RW1 to the RNeasy Mini spin column, followed by the addition of DNase I. The resulting RNA was evaluated for purity and concentration using a Nanodrop spectrophotometer from TECAN Reader For RNA sequencing, we used 2 μg of total RNA per sample concentration range 50-200 ng/µL, at least 10µL volume, checking the quality by RIN > 7, OD260/280 = 1,8-2,1 and OD260/230 >1,5. mRNA libraries were prepared by mRNA Seq (Poly A+ selection) using ERCC RNA Spike-In controls. Libraries were prepared using the Illumina® Stranded TruSeq® RNA sample preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. Library preparation started with 1000 ng total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (15 cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of the library were pooled. The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System. The pool was sequenced on an Illumina NovaSeq6000 sequencing instrument with a PE100 protocol aiming for 40 million clusters per sample.