Mouse breeding and husbandry
All experimental procedures related to the use of mice were approved by the Institutional Animal Care and Use Committee of the Allen Institute for Brain Science (AIBS) in accordance with NIH guidelines. Mice were housed in rooms with controlled temperature (21–22 °C) and humidity (40–51%) conditions at no more than five adult animals of the same sex per cage. Mice were provided food and water ad libitum and were maintained on a regular 14:10 h light–dark cycle. Mice were maintained on the C57BL/6J (RRID: IMSR_JAX:000664) background. We excluded any mice with anophthalmia or microphthalmia.
The presence of vaginal plugs was monitored at 12-h intervals (6:00 and 18:00). To collect embryos with accuracy to 0.5 days, only dams with visible plugs were used to obtain embryonic time points. For postnatal time points, births were recorded at 12-h intervals (6:00 and 18:00). Animal handling was reduced as much as possible until weaning at P21. At weaning, animals were separated from their mothers and opposite-sex siblings. Weaned mice were group-housed, kept separate from the opposite sex and maintained under normal housing conditions until dissection.
All animals used for data generation are listed in Supplementary Table 1. No statistical methods were used to predetermine sample sizes. In total we used 53 mice to collect scRNA-seq data from 913,297 cells across 35 time points between E11.5 and adulthood: embryonic day E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.0, E17.5, E18.0 and E18.5; postnatal day P0, P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P19, P20, P21, P23, P25 and P28; and adult stage P54–P68 (collectively simplified as P56). Brain dissections for all groups took place in the morning. From ages E11.5 to E12.5, we collected whole brain tissue, from ages E13.5 to E14.5, we collected the cerebrum and the brainstem, and from other ages we dissected the visual cortex (VIS). Tissue dissection was performed on about 300-µm coronal sections, and the VIS was identified and dissected out using the Allen CCFv3 reference atlas. The adult samples were taken from the ABC–WMB dataset by selecting scRNA-seq libraries with regions of interest (ROIs) of VIS-PTLp or VISp. For snMultiome data generation, we collected data from 331,831 nuclei from 28 mice across 13 time points: E15.5, E16.0, E17.0, E18.0, P0, P2, P4, P5, P8, P9, P11, P14 and P56. At embryonic time points, we dissected the cerebrum and the brainstem and at postnatal time points, we collected either the VIS or the isocortex.
In some cases, transgenic mice were used for fluorescence-positive cell isolation by FACS. To enrich neurons profiled by scRNA-seq, cells were isolated from the pan-neuronal Snap25-IRES2-Cre line (RRID: IMSR_JAX:023525) crossed to the Ai14-tdTomato reporter (RRID: IMSR_JAX:007914) (31 out of 53 mice, Supplementary Table 1).
Single-cell isolation
Single cells were isolated following a cell-isolation protocol developed at the AIBS71. The brain was dissected, submerged in artificial cerebrospinal fluid (ACSF), embedded in 2% agarose and sliced into 350-μm coronal sections on a compresstome (Precisionary Instruments). Block-face images were captured during slicing. ROIs were then microdissected from the slices and dissociated into single cells.
Dissected tissue pieces were digested with 30 U ml–1 papain (Worthington PAP2) in ACSF for 30 min at 30 °C. Owing to the short incubation period in a dry oven, we set the oven temperature to 35 °C to compensate for the indirect heat exchange, with a target solution temperature of 30 °C. Enzymatic digestion was quenched by exchanging the papain solution 3 times with quenching buffer (ACSF with 1% FBS and 0.2% BSA). Samples were incubated on ice for 5 min before trituration. The tissue pieces in the quenching buffer were triturated through a fire-polished pipette with a 600-µm-diameter opening approximately 20 times. The tissue pieces were allowed to settle and the supernatant, which now contained suspended single cells, was transferred to a new tube. Fresh quenching buffer was added to the settled tissue pieces, and trituration and supernatant transfer were repeated using 300 µm and 150 µm fire-polished pipettes. The single-cell suspension was passed through a 70 µm filter into a 15 ml conical tube with 500 µl high-BSA buffer (ACSF with 1% FBS and 1% BSA) at the bottom to help cushion the cells during centrifugation at 100g in a swinging-bucket centrifuge for 10 min. The supernatant was discarded, and the cell pellet was resuspended in the quenching buffer. We collected 547,092 cells without performing FACS. The concentration of the resuspended cells was quantified, and cells were immediately loaded onto a 10x Genomics Chromium controller.
To enrich neurons or live cells in some samples, cells were collected by FACS (BD Aria II running FACSdiva v.8 (RRID: SCR_001456)) using a 130 μm nozzle, following a FACS protocol developed at AIBS72. Cells were prepared for sorting by passing the suspension through a 70 µm filter and adding Hoechst or DAPI (to a final concentration of 2 ng ml–1). The sorting strategy with example images has been previously described72. We collected 30,833 calcein-positive and Hoechst-positive cells, 18,992 Hoechst-positive cells, 13,912 RFP-positive cells and 302,468 RFP-positive and Hoechst-positive cells (Extended Data Fig. 1d, Supplementary Table 1 and Supplementary Fig. 4). Around 30,000 cells were sorted within 10 min into a tube containing 500 µl quenching buffer. Each aliquot of sorted 30,000 cells was gently layered on top of 200 µl high-BSA buffer and immediately centrifuged at 230g for 10 min in a centrifuge with a swinging-bucket rotor (the high-BSA buffer at the bottom of the tube slows down the cells as they reach the bottom, which minimizes cell death). No pellet could be seen with this small number of cells, so we removed the supernatant and left behind 35 µl of buffer, in which we resuspended the cells. Immediate centrifugation and resuspension allowed the cells to be temporarily stored in a high-BSA buffer with minimal ACSF dilution. The resuspended cells were stored at 4 °C until all samples were collected, usually within 30 min. Samples from the same ROI were pooled, the cell concentration quantified and the samples immediately loaded onto a 10x Genomics Chromium controller.
Single-nucleus isolation
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