GSM6833358: V2; Rattus norvegicus; RNA-Seq
Rats were briefly anesthetized with isoflurane (Patterson Veterinary, Greenly, CO) and swiftly decapitated. The brains were then rapidly removed, and the hippocampi were dissected according to previously described methods (Lein 2004; Zeier 2011). The hippocampal regions DG was separated, placed in the tubes, and flash-frozen in liquid nitrogen. All samples were stored in at -80░C until processed. RNA was isolated from subregions of a hippocampus from a single hemisphere of each animal. RNA Isolation was performed according to methods previously described (Ianov 2016; Barter 2019). Briefly, RNA was isolated using the RNeasy Lipid Tissue Mini kit (Qiagen, catalog number 74804). Total RNA was treated with DNase using the RNase-Free DNase Set (Qiagen, catalog number 79254). Concentration of total RNA was assessed using the NanoDrop 2000 spectrophotometer (Thermo Fisher catalog number ND-2000) and the RNA integrity number (RIN) was quantified using the Agilent 2200 Tapestation system with High Sensitivity RNA Screen Tape (Agilent Technologies, Santa Clara, CA). Mean RNA integrity across all samples was 9.05 (SEM ▒ 0.02) with values ranging from 8.2 to 9.3. Poly-A-selection for mRNA was performed using 250 ng of isolated total RNA in the Dynabeads mRNA DIRECT Micro kit (Thermo Fisher, catalog number 61021). External RNA Controls Consortium (ERCC) RNA Spike-In Control Mixes were included in all isolated mRNA samples (Thermo Fisher, catalog number 4456740). Library preparation was performed using the Ion Total RNA-seq Kit v2 (Thermo Fisher, catalog number 4475936). Ion Xpress barcodes (Thermo Fisher, catalog number 4475485) were included with libraries for multiplex sequencing. In summary, isolated mRNA was fragmented with RNase III then ligated to the Ion Adapter Mix v2. RNA samples were then reverse transcribed. cDNA from each biological replicate was uniquely barcoded and amplified with 16 PCR cycles. Library concentration from each sample was quantified using the Qubit dsDNA HS Assay (Thermo Fisher, catalog number Q32851). DNA fragment size distribution was subsequently evaluated using High Sensitivity D1000 ScreenTape in the 2200 Tapestation system (Agilent Technologies, Santa Clara, CA).   Templates were prepared using the Ion Chef system (Thermo Fisher, catalog number 4484177) and sequencing was performed using the Ion Proton system (Thermo Fisher, catalog number 4476610) or Ion GeneStudio S5 system (Thermo Fisher, catalog number A38194).