Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA-seq: RNA from 3 AL and 3 DR animals per time point was isolated from 30 mg of liver tissue using Trizol Reagent (ThermoFisher) according to the manufacturer’s instructions, followed by DNase treatment with the TURBO DNA-free Kit (ThermoFisher). RNA integrity was analyzed using the Agilent TapeStation System. BS-seq: DNA of 3 AL and 3 DR animals per timepoint was isolated from 30 mg of liver tissue using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA-seq: TruSeq RNA-seq library preparation was conducted as previously described (Sultan, et al., 2012) using 3 µg of total RNA as input and rRNA depletion. Barcoded libraries were sequenced with 2x40 mio, 100 bp paired-end reads on an Illumina HiSeq2500. BS-seq: Around 400 ng genomic DNA was used as input for BS-Seq library generation. DNA was sonicated, and end repair and A-tailing were performed using the NEB Next kit according to the manufacturers’ instructions. Illumina’s Early Access Methylation Adaptor Oligo Kit was used for adaptor ligation. Adaptor-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturer’s instructions for the one-step protocol. Bisulfite-treated DNA was amplified using PfuTurbo Cx Hotstart DNA Polymerase from Agilent Technologies with 14–18 cycles depending on input amount. Size selection was performed by gel extraction for DNA fragments between 200 bp and 250 bp, as adapted from (Seisenberger et al., 2012). Libraries of young samples were sequenced using the paired-end protocol. Old sample libraries were sequenced using single-end protocol and a subsequent re-run using a paired-end protocol to increase coverage.