GSM9059629: snRNA_HC_rep2; Mus musculus; RNA-Seq
Mice were deeply anesthetized with isoflurane, decapitated and brains were placed in a stainless-steel brain matrix on ice to cut 1mm sections of anterior to posterior hippocampus. Sections were transferred into ice-cold PBS and Cornu Ammonis (CA)1, CA3 and Dentate Gyrus (DG) separated under a dissection microscope. Tissue was frozen in liquid nitrogen and stored at -80° for further processing and RNA extraction. For bulk RNA-seq,tissue was resuspended in 500 µL TRIzol reagent (Life Technologies), followed by trituration with a 26G needle. RNA was chloroform extracted, an equal volume of 100% ethanol was added, and RNA was purified using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instruction, including the on-column DNase digest.  For single-nuclei multiomic-sequencing whole hippocampi were dissected on an ice-cold metal block and immediately processed for nuclei preparation. For bulk RNA-seq, RNA concentration was assessed by Nanodrop and RNA-seq libraries were prepared from 10 ng of total RNA using the SMARTer Stranded Total RNA-seq Pico Input Mammalian V2 kit (Takara Bio) according to the manufacturer's instructions. For snMultiom-seq, freshly dissected hippocampi were dissociated, nuclei were prepared then concentrated in 1X nucleus buffer from 10X Genomics.  1µl of nuclei was stained with Trypan Blue (Invitrogen, T10282) and manually counted. In total, 16,000–20,000 nuclei were used for the tagmentation reaction and 10x Chromium controller loading. The DNA and RNA libraries were generated according to the manufacturer's recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression).