GSM3619371: NRNA-8; Mus musculus; RNA-Seq
Ventral hippocampus was dissected from the brain, snap frozen in liquid nitrogen, and total nuclei were extracted using sucrose gradient centrifugation. Neuronal (NeuN+) nuclei were isolated by fluorescence-activated nuclei sorting  with anti-NeuN antibody conjugated to AlexaFluor 488 on a FACSAria instrument. Detailed protocol for total nuclei isolation and purification of neuronal nuclei is provided in Kundakovic et al, 2017 (PMID:27113501). Per each biological replicate, 250,000 neuronal nuclei were directly sorted into Trizol LS reagent. After the addition of chloroform, the aqueous phase was recovered and total nuclear RNA was further isolated and purified using the miRNeasy Micro kit reagents and the RNeasy MinElute spin column. Nuclear RNA isolated from sorted neuronal nuclei (16-26 ng/sample) was used to construct cDNA sequencing library using the KAPA RNA HyperPrep Kit with RiboErase (KK8560, KAPA Biosystems), following general manufacturer's instructions. Briefly, rRNA was first depleted from total nuclear RNA by hybridization of complementary DNA oligonucleotides followed by treatment with RNase H and DNase. rRNA-depleted nuclear RNA was fragmented at 94°C for 5 minutes in the presence of Mg2+, followed by the first and second strand cDNA synthesis and A-tailing. Following the adapter ligation with the KAPA single-indexed adapters (KK8701), each library was amplified using 13 PCR cycles. After a bead-based cleanup, the library quality was examined using Bioanalyzer High-Sensitivity DNA Assay and the libraries were quantified by Qubit HS DNA kit prior to sequencing.
GEO Accession
GSM3619371