GSM5941117: 8_AD_hippocampus; Mus musculus; RNA-Seq
The brain tissue was homogenized in TRIzol® Reagent (Ambion) using a Precellys homogenizer (Bertin Instruments) and extracted according to the TRIzol® Reagent protocol, with an additional phenol/chloroform purification step and the final isopropanol precipitation. For the total RNA sequencing we used 1,000 ng of total RNA; rRNA was depleted using a RNase H-based protocol. We mixed total RNA with 1 μg of a DNA oligonucleotide pool comprising a 50-nt long oligonucleotide mix covering the reverse complement of the entire length of each human rRNA (28S rRNA, 18S rRNA, 16S rRNA, 5.8S rRNA, 5S rRNA, 12S rRNA), incubated with 1U of RNase H (Hybridase Thermostable RNase H, Epicentre), purified using RNA Cleanup XP beads (Agencourt), DNase treated using TURBO DNase rigorous treatment protocol (Thermo Fisher Scientific), and purified again with RNA Cleanup XP beads. We fragmented the rRNA-depleted RNA samples and processed them into strand-specific cDNA libraries using TruSeq Stranded Total LT Sample Prep Kit (Illumina) and then sequenced them on NextSeq 500, High Output Kit, 1 x 150 cycles.