RNAseq of mus musculus: male treated with rapamycin from P30 to P34
Libraries were prepared from the extracted RNAs using the QuantSeq 3mRNA-Seq Library Prep Kit-FWD (Lexogen, Vienna, Austria) using 1 g of RNA per library and following the manufacturers instructions. We modified the standard protocol by adding Unique Molecular Identifiers (UMI) during the second strand synthesis step. Indices from the Lexogen i7 6nt Index Set and i5 6nt Dual Indexing Add-on Kits (Cat. No. 044.96 and 047.96, Lexogen) were used, and 15 cycles of library amplification were performed. Libraries were eluted in 30 L of the kits Elution Buffer. The double-stranded DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher), ranging from 3 to 12 ng/L. The molar concentration of cDNA molecules in the individual libraries was calculated from the double-stranded DNA concentration and the single library average size (determined on a PerkinElmer Labchip GX). An equal number of cDNA molecules from each library were pooled and the final pool was purified once more in order to remove any free primer and prevent index-hopping. The pooled libraries were sequenced in a Novaseq 6000 instrument (Illumina, San Diego, CA) on an SP flowcell, producing 900M single reads 100nt.