GSM5133910: 10684_BAT; Mus musculus; RNA-Seq
Tissue was frozen at -80°C until the RNA was extracted with DNase digestion using the RNeasyR Lipid Tissue Mini Kit and RNase-Free DNase Set (Qiagen), following the user's manual. RNA integrity was detected with the Agilent RNA 6000 Nano Kit (5067-1511, Agilent Technologies) and the Agilent 2100 Bioanalyzer. RNA libraries were prepared using the Illumina® TruSeq® mRNA stranded sample preparation Kit. Library preparation started with 1µg 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 is 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 (14 PCR cycles) to create the final cDNA libraries. After library validation and quantification (Agilent tape station), equimolar amounts of library were pooled. The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.