Nature - USA (2020-01-16)

2

nature research | reporting summary

October 2018

Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size No sample size pre-determination was performed. The largest number of cells that could be practically sequenced from an mice were done in

all conditions. This study does not claim to saturate the discovery of all possible cell types nor saturate the detection of all possible difference.

However, given the number of cell sequenced, we were able to confidently characterize cell types based on previously published literature as

well as generate new Cre drivers that labeled morphologically homogenous cell types. This suggests that the number of cells sequenced is

sufficient to support the claims of the study.

Data exclusions Individual cells with high mitochondrial read count or low gene discovery were excluded from further analysis and predetermined based on

standards previously determined - a common practice for single cell studies. The number of cells excluded were roughly equal across all time

points and experimental conditions.

Replication All replication attempts were successful by our best efforts. To validate scRNA-seq experiments we performed strategic in situ validation. To

validate the faithfulness of the mouse reporter lines, we performed double in situs with the reporter and the endogenous gene driving Cre to

confirm that the cell type of interest was properly labeled.

Randomization Animals used for behavior were litter mates (test and control groups) and randomly selected. All time points for scRNA-seq were performed in

random orders.

Blinding Blinding was not possible in our study as all conditions were sequenced in parallel so the investigator could not be aware of the results until

after completion of the experiment.

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Antibodies

Antibodies used Rabbit Anti-NeuN, Millipore: MAB377, 1:1000. Goat Anti-mCherry/tdtomato, CederLane: AB0040-200, 1:1000, Chicken Anti-GFP,

Aves: GFP-1020, 1:1000, Rabbit Anti-GFP, ThermoFisher: A6455, 1:1000, Rabbit Anti-CGRP, Immunostar: 24112, 1:1000.

Secondary antibodies included Alexa 488 or 546 conjugated goat anti-rabbit antibodies, goat anti-chicken antibodies, All

secondary antibodies were purchased from Life Technologies except for Alexa 488 conjugated donkey anti-chicken antibodies,

which was purchased from Jackson ImmunoResearch, and all were used at 1:500 dilution

Validation All antibodies were validated in previous studies from our lab and others.

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) 293T cells (ATCC)

Authentication Cells lines were not authenticated in this study - they were only used as a tool to generate adeno-associated virus