Nature - USA (2020-01-23)

6

nature research | reporting summary

October 2018

Flow Cytometry

Plots

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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 Spleens and lymph node cells from mice were harvested and homogenized. Cells were suspended in FACS buffer(2% FBS in PBS)

and cell suspensions were filtered through 70um cell strainer. The suspension was washed with cold FACS buffer and centrifuged

at 500g for 5min.

Instrument LSRFortessa, FACS Aria III

Software BD FACS Diva 8.0.1 software was used for data collection and FlowJo v.10 was used for data analysis.

Cell population abundance For the spleen and lymph node cells, there are~25% of CD4+ T cell, ~20% of CD8+ T cell in single alive lymphocytes. For CD4+ T

cells, there are ~85% of CD62L+CD44- naïve T cells.

For the spleen lymphocytes, there are~20% of CD3+ T cell and ~50% of B220+ B cell in WT mouse, ~10% of CD3+ T cell in CKO

mouse.

For flow sorting, post-sort cells were analyzed on BD Aria III and the purity was at least 95%.

For the cell cycle, there are ~65% of G1/G0 phase cell, ~25% of S phase cell and ~10% of M phase cell in WT group, ~80% of G1/

G0 phase cell, ~10% of S phase cell and ~10% of M phase cell in H2A.Z KO group.

For flow sorting, post-sort cells were analyzed on BD Aria III and the purity was at least 95%.

Gating strategy I. For T cell analysis and sorting

I-a. For spleen lymphocytes, T cells were gated with CD3 and B cell gated with B220.

I-b. For sorting naïve T cells, single alive lymphocytes were first gated with CD4 and CD8, then naïve T cells were gated with CD44

and CD62L, CD44 low and CD62L high cells were resident naïve T cells.

II. For CFSE analysis

Divied cells: single alive lymphocytes were gated with CFSE+, and CFSE diluted cells are divied cells.

III. For cell cycle analysis

Single alive lymphocytes were gated with Brdu and PI. Brdu low and PI low cells were resident G1/G0 phase cells; Brdu high and

PI middle cells were resident S phase cell cells; Brdu low and PI high cells were resident M phase cell cells.

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