Single-nucleus RNA-seq of Mus musculus: threat conditioned adult female proestrus lateral septum
Single-nucleus multiomic sequencing was performed on lateral septum (LS) tissue from adult C57BL/6J female mice in proestrus. Animals were randomly assigned to threat-conditioned or threat-naive groups (n=4 per group). Threat conditioning consisted of six co-terminating pairings of a 20 s auditory conditioned stimulus (5 kHz, 75 dB) with a 2 s, 0.5 mA foot shock following a 240 s baseline period. Inter-trial intervals were 100 s. Naive controls remained in the home cage. Five minutes after exiting the conditioning chamber, mice were anesthetized and euthanized by rapid decapitation. Brains were removed, sectioned at 1 mm, and 1.25 mm punches of the lateral septum were collected, snap-frozen, and stored at 80 C. LS punches were pooled by experimental group prior to nuclei isolation. Nuclei were isolated using detergent-based lysis, filtered (35 m), washed, and resuspended in Hibernate AB. Nuclei were stained with 7-AAD and singlets were sorted by flow cytometry. Suspensions were processed using the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression platform. Gene expression libraries were sequenced on an Illumina NovaSeq 6000 using v1.5 chemistry (28  10  10  90 cycles) targeting >50,000 reads per nucleus.