GSM7968397: Male_eGFP_3; Mus musculus; RNA-Seq
Animals were sacrificed by cervical dislocation and bilateral ventral hippocampi were rapidly dissected then flash frozen in liquid nitrogen. 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. Nuclei were direclty sorted into TRIzol. Following sorting of nuclei, chloroform was added to the sample and the aqueous phase was recovered. nucRNA was then isolated and purified using the RNeasy Micro kit (Qiagen). The quality of nucRNA was assessed using the Fragment Analyzer (Agilent), with each sample having RQN>7. nucRNA quantity was determined using the Qubit RNA High-Sensitivity assay (Thermo Fisher Scientific, Q32852). All purified nucRNA (9-20 ng/sample) was used to construct cDNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KK8560, KAPA Biosystems), following the manufacturer's instructions. rRNA was depleted from nucRNA samples using hybridization of DNA oligonucleotides complementary to rRNA followed by RNase H and DNase treatment. The efficiency of rRNA depletion was confirmed using the Bioanalyzer (Agilent) and with qRT-PCR amplification of the 28S rRNA transcript before and after RiboErase treatment using the following primers: Forward, 5'-CCCATATCCGCAGCAGGTC-3'; Reverse, 5'-CCAGCCCTTAGAGCCAATCC-3';. rRNA-depleted nucRNA was then fragmented at 94°C for 5 minutes in the presence of Mg2+, followed by first and second strand cDNA synthesis and A-tailing. After ligation of KAPA dual-indexed adapters (KK8722), each library was amplified using 13-14 PCR cycles. Following a bead-based clean-up, the length of the libraries (365bp, on average) and their quality was assessed using the Bioanalyzer High-Sensitivity DNA Assay. The libraries were quantified with the Qubit HS DNA kit (Life Technologies, Q32851) and by qPCR (KAPA Biosystems, KK4873) prior to sequencing.