GSM8842365: 1105; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane and sacrificed by decapitation. Brains were immediately extracted and placed in ice-cold oxygenated artificial cerebro-spinal fluid (ACSF) with an osmolarity of 300 mOsm and pH at 7.4. Coronal sections of the brain spanning the antero-posterior localization of the claustrum were cut at a thickness of 300 μm using a vibrating-blade microtome. Immediately after sectioning, the claustrum and adjacent brain regions were microdissected under a stereomicroscope. Tissues from each mouse were processed individually, with no pooling across different animals. Tissue dissociations were performed using the Papain Dissociation System (ref. LK003150; Worthington® Biochemical Corporation) according to the manufacturer's protocol with slight modifications, using EBSS and medium with serum solutions adapted from Saxena et al. (DOI: 10.2144/0000113878). To ensure the exclusive isolation of live nucleated cells, cell suspensions were incubated with 2 μg/ml of Hoechst 33342 (a UV fluorescent adenine-thymine binding dye; ref. H1399, Life Technologies) at 37°C for 15 min. In order to exclude dead cells, 1 μM of DRAQ7 (a far-red fluorescent DNA intercalating dye; ref. DR71000, BioStatus) was added to the cell suspensions before fluorescence activated cell sorting (FACS). Between 11'000-25'000 Hoechst+/DRAQ7- cells were collected from each sample. Single-cell capture, reverse transcription, cDNA amplification, and library preparation were performed using the 10x Genomics Chromium Controller instrument and the Single Cell 3′ v3.1 reagent kit with dual indexes, following the manufacturer's protocol (CG000315 Rev B User Guide). Cells were loaded on the 10x Genomics Chromium Controller instrument at a concentration of 400 cells/μl, and the targeted cell recovery was adjusted for each sample based on the number of sorted cells. For a detailed description of the protocol, please refer to the methods section of Fodoulian et al. (2024) bioRxiv (https://doi.org/10.1101/2024.11.05.622016). Libraries from mice of the same age were pooled and each loaded at 2 nM on 4 lanes for clustering on a paired-end Illumina Flow Cell using the HiSeq 4000 PE Cluster Kit (ref. PE-410-1001; Illumina) and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq 4000 SBS Kit (ref. FC-410-1001; Illumina) chemistry. Read 1 consisted of 28 bases, while read 2 was either 90 or 100 bases. An average of 5'171 cells were sequenced with 42'199 mean reads, 1'125 median genes, and 1'999 median UMI counts per cell.