Uncovering the Molecular Logic of Cortical Wiring between Neuronal subtypes Across Development Through Ligand–Receptor Inference
Uncovering the Molecular Logic of Cortical Wiring between Neuronal subtypes Across Development Through Ligand–Receptor Inference
Animals: Mice (Mus musculus) were group-housed (2–5 mice per cage) with same-sex littermates under a 12-hour light-dark cycle, with ad libitum access to food and water. Animals were bred and maintained on a mixed SVeV-129/C57BL/6N genetic 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). Single-Cell Isolation: Mice brains were dissected in ice-cold, carbogen-bubbled (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF). The ACSF composition was: 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 kynurenic acid (0.567 g/L). Brains were sliced into 300 µm coronal sections (P0 and P2 mice) or 500 µm coronal sections (P5, P8, and P30 mice) using a vibratome (Leica). The somatosensory cortex was dissected under a binocular microscope. Single-Nuclei Isolation: The somatosensory cortex was dissected as described for single-cell isolation. Dissected cortices were immediately transferred into 500 µL of Hibernate™-E Medium (#A12476-01) and flash-frozen for 3 minutes in isopentane pre-cooled to -80°C. Samples were stored at -80°C for long-term preservation. Tissue samples for those datasets underwent enzymatic digestion with pronase (Streptomyces griseus, 1 mg/mL) for 25 minutes at room temperature. Cells were dissociated and triturated into a single-cell suspension using three glass Pasteur pipettes of decreasing diameters in ACSF supplemented with 1% fetal calf serum (FCS) and DNase (1 µL/10 mL). For P30 datasets, enzymatic digestion and cell dissociation followed the Worthington Papain Dissociation System manufacturer's instructions  Enzymatic digestion was processed by using pronase (Septomyces Argeus at 1 mg/mL) during 25 min at room temperature (RT) for P0 to P8 datasets. Cells were dissociated and triturated into single cell suspension in a solution consisted of ACSF, 1% FCS and DNAse (1µl/10mL). Trituration was carried out by using 3 glass pasteur pipets prepared at 3 different diameters. For P30 datasets, we used the Worthington Papain Dissociation System to carry out the enzymatic digestion and the cell dissociation following the manufacturer instructions. Single-cell suspensions were loaded onto a Chromium Next GEM Chip G using the Chromium Next GEM Single Cell 3' Reagent Kits (10x Genomics), following the manufacturer's protocol. Individual cells or nuclei were encapsulated into Gel Beads-in-Emulsion (GEMs), where barcoding and reverse transcription (GEM-RT) occurred to generate barcoded full-length cDNA.  After GEM-RT incubation, emulsions were broken, and the barcoded cDNA was recovered and amplified via PCR with SPRIselect bead-based cleanups. The amplified cDNA was enzymatically fragmented, followed by end repair, A-tailing, adaptor ligation, and sample index PCR to construct final 3' gene expression sequencing libraries.  Constructed libraries were sequenced on an Illumina HiSeq 4000 platform. The final libraries incorporate Illumina P5 and P7 adapters, with Read 1 capturing the 10x barcode and UMI sequences and Read 2 sequencing the cDNA fragment for gene expression analysis.