Single cell isolation

We adapted a previously described procedure to isolate fluorescently labeled neurons from the mouse brain^{5, 65}. Individual adult male mice (P56 ± 3) were anesthetized in an isoflurane chamber, decapitated, and the brain was immediately removed and submerged in fresh ice-cold artificial cerebrospinal fluid (ACSF) containing NaCl (126 mM), NaHCO₃ (20 mM), dextrose (20 mM), KCl (3 mM), NaH₂PO₄ (1.25 mM), CaCl₂ (2 mM), MgCl₂ (2 mM), DL-AP5 sodium salt (50 µM), DNQX (20 µM), and tetrodotoxin (0.1 µM), bubbled with a carbogen gas (95% O₂ and 5% CO₂)_. The brain was sectioned on a vibratome (Leica VT1000S) on ice, and each slice (300–400 µm) was immediately transferred to an ACSF bath at room temperature. After the brain slicing was complete (not more than 15 minutes), individual slices of interest were transferred to a small Petri dish containing bubbled room temperature ACSF. The regions of interest (all layers of VISp or specific layers of VISp) were microdissected under a fluorescence dissecting microscope, and the slices before and after dissection were imaged to later examine the location of the microdissected tissue and confirm its location within VISp. The dissected tissue pieces were transferred to a microcentrifuge tube and treated with 1 mg/ml pronase (Sigma, Cat#P6911-1G) in carbogen-bubbled ACSF for 70 minutes at room temperature without mixing in a closed tube. After incubation, with the tissue pieces sitting at the bottom of the tube, the pronase solution was pipetted out of the tube and exchanged with cold ACSF containing 1% fetal bovine serum. The tissue pieces were dissociated into single cells by gentle trituration through Pasteur pipettes with polished tip openings of 600-µm, 300-µm, and 150-µm diameter³⁷.

Single cells were isolated by FACS into individual wells of 96-well plates or 8-well PCR strips containing 2.275 µl of Dilution Buffer (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936), 0.125 µl RNase inhibitor (SMARTer kit), and 0.1 µl of 1:1,000,000 diluted RNA spike-in RNAs (ERCC RNA Spike-In Mix 1, Life Technologies Cat#4456740). Sorting was performed on a BD FACSAriaII SORP using a 130 µm nozzle, a sheath pressure of 10 psi, and in the single cell sorting mode. To exclude dead cells, DAPI (DAPI*2HCl, Life Technologies Cat#D1306) was added to the single cell suspension to the final concentration of 2 ng/ml. FACS populations were chosen to select cells with low DAPI and high tdT fluorescence. Accuracy of single cell sorting was evaluated as described in Supplementary Fig. 2a, and confirmed post-hoc by observing dramatically higher expression of tdT mRNA in tdT⁺ than in tdT⁻ cells (Supplementary Fig. 2c). In some cases, we also selected cells that have low DAPI and low tdT fluorescence, in order to capture tdT⁻ cells from a sample. To collect all cells in an unbiased manner, we selected all cells with low DAPI fluorescence, regardless of their tdT fluorescence level. Sorted cells were frozen immediately on dry ice and stored at −80 °C.

In total we used 72 animals, with at least two animals per Cre line in most cases. One animal each was used for the Chat-IRES-Cre, Tac1-IRES2-Cre, Gad2-IRES-Cre, and Slc17a6-IRES-Cre lines. The 72 animals were used for 55 specific dissection conditions (unique combination of Cre, layer dissection, and tdT labeling, Supplementary Table 3), with 34 conditions corresponding to one animal each, 13 conditions corresponding to two animals, and five conditions corresponding to three animals, two conditions corresponding to four animals, and one condition corresponding to five animals.