Nature - USA (2019-07-18)

3

nature research | reporting summary

October 2018

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) 293T (ATCC CRL-3216)

Authentication Cell lines obtained directly from ATCC. No cell line authentication performed.

Mycoplasma contamination Cell lines were not tested for mycoplasma

Commonly misidentified lines

(See ICLAC register)

The cell line used is not listed in the ICLAC database.

Animals and other organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals Mus musculus, B6, sex-matched male and female, 6-8 weeks old

Wild animals n/a

Field-collected samples n/a

Ethics oversight All mice required for this study were housed and maintained under specific-pathogen-free conditions in the animal facility of the

Yale University School of Medicine, and all corresponding animal protocols were approved by the Institutional Animal Care and

Use Committee (IACUC) of Yale University. This study was conducted in compliance with all relevant ethical regulations.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.

Methodology

Sample preparation Cells were harvested, washed at least once with PBS, and stained with antibodies targeting surface antigens in 2%FBS 1mM EDTA

in PBS for 30 minutes on ice. For analysis of intracellular cytokine staining, cells were then fixed and permeabilized with fixation/

permeabilization solution (BD Cytofix/Cytoperm Cat#554714) at 4C for 20 minutes. For nuclear transcription factor staining,

cells were fixed and permeabilized with eBioscience Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and

Diluent (Cat #00-5521-00). Cells were then washed three times with 1X permeabilization buffer (eBioscience Permeabilization

Buffer Cat #00-8333-56) and stained for intracellular antigens for 1 hour on ice in 1X permeabilization buffer. Cells were then

washed two times before proceeding to cytometry analysis. For cells not transduced with retrovirus, cells were labeled with

LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Cat# L34965) per the manufactures protocol before surface staining.

Further details regarding specific staining protocols and antibodies used can be found in the methods section of the manuscript.

Cells were harvested, washed at least once with PBS, and stained with antibodies targeting surface antigens in 2%FBS 1mM EDTA

in PBS for 30 minutes on ice. For analysis of intracellular cytokine staining, cells were then fixed and permeabilized with fixation/

permeabilization solution (BD Cytofix/Cytoperm Cat#554714) at 4C for 20 minutes. For nuclear transcription factor staining,

cells were fixed and permeabilized with eBioscience Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and

Diluent (Cat #00-5521-00). Cells were then washed three times with 1X permeabilization buffer (eBioscience Permeabilization

Buffer Cat #00-8333-56) and stained for intracellular antigens for 1 hour on ice in 1X permeabilization buffer. Cells were then

washed two times before proceeding to cytometry analysis. For cells not transduced with retrovirus, cells were labeled with

LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Cat# L34965) per the manufactures protocol before surface staining.

Further details regarding specific staining protocols and antibodies used can be found in the methods section of the manuscript.

Instrument BD LSRII custom order product

Software BD FACSDiva Software was used to collect raw data files from all flow cytometry experiments. All resultant data files were

analyzed using FlowJo version 8 or newer.

Cell population abundance Cells were purified for three replicate screens using a BD FACS Aria as described in methods. Due to limited cell numbers, all

resultant cells were processed for gDNA isolation and sequencing.