title: GSM5097085: HD36_MWM_water; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from dorsal hippocampal tissues using TRIzol reagent (Invitrogen) (n=4/group). Freshly dissected tissue was chopped, homogenized in 300 μL of TRIzol reagent, and frozen (20 minutes at -80°C), followed by 3-minutes centrifugation at 14000xg before chloroform/isoamyl extraction. The supernatant was used to precipitate RNA using isopropanol and RNase-free glycogen (30 minutes at 4°C). The pellet was washed once with 70% ethanol and resuspended in Milli-Q water. A new RNA precipitation was performed with 100% ethanol and 3 M NaOAC (overnight at -20°C). After two further 70% ethanol washes, pellet was air-dried and resuspended in 30 μL nuclease-free Milli-Q water, heated 6 minutes at 50°C, and RNA quantification was performed. RNA-Seq libraries were generated from 500 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.