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nature research | reporting summary

April 2018

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 Bone marrow cells were collected by flushing femora and tibias stems of both lower limbs, spleen cells were collected by

mincing the whole spleen with frosted slides, treating with ACK buffer to get rid of red blood cells unless performing peripheral

whole blood compartment studies, and filtered with Falcon 70 micrometer nylon cell strainer. E14.5 fetal liver cells were

collected by separating fetal livers from E14.5 feti, and further mincing the livers with frosted slides. All population staining was

conducted directly on live cells, and translation studies were conducted by labeling live cells with O-Propargyl-puromycin in RPMI

1640 medium containing 15% FBS for 1 hr, and then samples were fixed with 4% paraformaldehyde, permeabilized with 0.1%

saponin before conducting CLICK-IT assay according to manufacture protocol.

Instrument All FACS data was collected either on LSR II flow cytometry system from BD, or on FACSCalibur flow cytometry system from BD.

Software FACS data conducted on LSR II was collected using BD FACSDiva software, and FACS data conducted on FACSCalibur was

collected using BD CellQuest Pro. All FACS data was further gated and analyzed using Flowjo V10.

Cell population abundance No cell sorting was conducted in this study.

Gating strategy All gating were conducted following well accepted gating strategies of corresponding antibodies staining from published studies^

before. On preliminary FSC/SSC plot, gates were set on gaps between debris, dead cells and live cells. And all live cells were

included in further analysis.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.