title: GSM8503823: MNs.female.post-exp. # 2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from the samples using the Trizol Reagent (Invitrogen Life Technologies) followed by RNA isolation according to the manufacturer's protocol. Three micrograms of RNA were used as input material for the RNA sample  preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA  was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was  carried out using divalent cations under elevated temperature in an Illumina proprietary  fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and  Super Script II. Second strand cDNA synthesis was subsequently performed using DNA  Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via  exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′  ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for  hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library  fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA).  DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP  system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100  system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform  (Illumina).