Mice were euthanized under anesthesia and the hippocampi were quickly dissected over ice. Total RNA was extracted using miRNeasy minikit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Isolated RNA was eluted in RNase-free water and submitted to the Genomic and RNA Profiling Core at Baylor College of Medicine. Sample quality checks using the NanoDrop spectrophotometer and Agilent Bioanalyzer 2100 were conducted. A double-stranded DNA library was created using 250 ng of total RNA (measured by picogreen), preparing the fragments for hybridization onto a flow-cell. First, cDNA was created using the fragmented 3’ poly(A) selected portion of total RNA and random primers. During second strand synthesis, dTTP is replaced with dUTP which quenches the second strand during amplification, thereby achieving strand specificity. Libraries were created from the cDNA by first blunt ending the fragments, attaching an adenosine to the 3’ end and finally ligating unique adapters to the ends. The ligated products were then amplified using 15 cycles of PCR. The resulting libraries were quantitated using the NanoDrop spectrophotometer and fragment size assessed with the Agilent Bioanalyzer. A qPCR quantitation was performed on the libraries to determine the concentration of adapter ligated fragments using Applied Biosystems ViiA7 Real-Time PCR System and a KAPA Library Quant Kit. Using the concentration from the ViiA7 qPCR machine, 21pM of library was loaded onto a flowcell and amplified by bridge amplification using the Illumina cBot machine. A paired-end 100 cycle run was used to sequence the flow-cell on a HiSeq Sequencing System.
GEO Accession
GSM1831215