title: GSM9557191: dorsal MD nuclei, MD-projecting, male; Mus musculus; RNA-Seq
library_construction_protocol: H2B-TRAP mice aged 3-4 months received bilateral stereotaxic injections of AAVrg-hSyn-Cre into either the DMS or MD. After 2 weeks of recovery, mice were sacrificed between 9:00 am – 12:00 pm by rapid decapitation immediately after removal from the home cage. Brains were extracted and chilled in DMEM/F12 media slush (Thermo Fisher Scientific). Brains were then placed ventral side up in a chilled stainless steel brain matrix (Stoelting, Wood Dale, IL) and 1 mm coronal sections of the PFC were collected. Tissue from the PFC was placed in chilled RNAprotect solution (QIAGEN, Aarhus, Denmark) and then microdissected under a fluorescent stereoscope. During dissections tissue was pooled according to projection target (DMS or MD) and PFC subregion (dorsal or ventral), with male and female samples pooled separately. All pooled samples were incubated overnight at 4°C in RNAprotect® before being removed and stored dry at -80°C. Each sample underwent nuclear dissociation and purification procedures before being incubated with NucBlue (Thermo fisher scientific). After incubation, nuclei were flow sorted together into RT Reagent B (10x Genomics) using a  Cytek Aurora CS cell sorter with a 70 μm nozzle to remove poor quality nuclei and count the number of nuclei per sample. Sorted nuclei were immediately loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. Single Cell 3' v3.1 chemistry was used to process all samples. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). Gene expression libraries were multiplexed and sequenced on a NovaSeq 6000 (Illumina) sequencer using a 100-cycle kit with  the following read structure: Read 1 = 28 cycles followed by Read 2 = 90 cycles.