Single-nucleus data for Uncovering the Molecular Logic of Cortical Wiring between Neuronal subtypes Across Development Through Ligand–Receptor Inference
Mice (Mus musculus) were group-housed (2–5 mice per cage) with same-sex littermates on a 12-hour light-dark cycle, with ad libitum access to food and water. They were bred and maintained on a mixed SVeV-129/C57BL/6N background. All animal experiments were conducted in accordance with European Communities Council Directives and approved by French ethical committees (Comité d'Éthique pour l'expérimentation animale no. 14; permission number: 62-12112012, Apafis #21683-2019073011285386v4). For sample collection, mice brains were dissected while submerged in ice-cold, carbogen-bubbled (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF). The ACSF composition was as follows: NaCl (7.32 g/L), KCl (0.26 g/L), NaH₂PO₄·H₂O (0.165 g/L), CaCl₂·2H₂O (0.438 g/L), MgCl₂·6H₂O (0.264 g/L), D(+)-Glucose (1.98 g/L), NaHCO₃ (2.1 g/L), and acid kynurenic (0.567 g/L). The somatosensory cortex was dissected under a binocular loop. For single-nucleus isolation, the dissected somatosensory cortex was immediately transferred into 500 µL of Hibernate™-E Medium (#A12476-01) and frozen for 3 minutes in isopentane pre-cooled to -80°C. Samples were stored at -80°C for long-term preservation. After conservation, the medium was removed, and 500 µL of chilled 0.1X NP40 Lysis Buffer was added. The tissue was homogenized using a Pellet Pestle (15 strokes) and incubated on ice for 5 minutes. The suspension was pipette-mixed 10 times with a wide-bore tip and incubated for an additional 10 minutes on ice. After lysis, 500 µL of chilled wash buffer was added, and the mixture was pipette-mixed 5 times with a regular-bore tip. The suspension was filtered through a 30 µm cell strainer to remove debris, transferred to a 1.5 mL tube, and centrifuged at 950 × g for 10 minutes at 4°C. The supernatant was carefully removed, and the nuclei pellet was retained for downstream analysis.  Nuclei suspensions were prepared according to 10x Genomics recommendations. GEM generation and barcoding were performed using the Chromium Next GEM Single Cell 3' Reagent Kits, following the manufacturer's protocol. Single-nucleus suspensions were loaded onto a Chromium Next GEM Chip G, and GEMs were generated using the Chromium Controller. After GEM-RT incubation, cDNA was recovered, cleaned up, and amplified using SPRIselect bead-based purification. The amplified cDNA underwent fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR to construct the final 3' gene expression libraries.