Additional file 1 of An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion
Creators: name: Zhou, Kai
name: Han, Jinming
name: Lund, Harald
name: Boggavarapu, Nageswara Rao
name: Lauschke, Volker M
name: Goto, Shinobu
name: Cheng, Huaitao
name: Wang, Yuyu
name: Tachi, Asuka
name: Xie, Cuicui
name: Zhu, Keying
name: Sun, Ying
name: Osman, Ahmed M.
name: Liang, Dong
name: Han, Wei
name: Gemzell-Danielsson, Kristina
name: Betsholtz, Christer
name: Zhang, Xing-Mei
name: Zhu, Changlian
name: Enge, Martin
name: Joseph, Bertrand
name: Harris, Robert A.
name: Blomgren, Klas
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Additional data related to Fig. 1. A Representative 8 sections of Hoechst staining of Cx3cr1CreER-Eyfp/wt mouse brain sequential sagittal slices, the numbers representing the location and number of EYFP− microglia in that location, each color represents one mouse, n = 6. Figure S2. Morphology analysis. A The process to prepare topological skeleton from the original photomicrographs. The full-size maximum intensity projection images (top) were processed into binary images (middle) and then skeletonized (bottom) following the ImageJ plugin protocol. Cropped images (right) from the original full-size images (left) were shown to improve the visualization. Scale bar, 50 μm. B The Analyze Skeleton plugin was applied, and skeletonized endpoints were tagged purple, the slab is orange, and the junction is pink. The tagged data are summarized as total length (sum of endpoints, slab, and junction) and the total number of endpoints. C The process area is represented as the convex hull area by connecting the process ends using the ImageJ polygon tool. Figure S3. Additional data related to Fig. 1. A Gating strategies of flow cytometry analyses. B EYFP− microglia ratio of total microglia at 3 weeks, 4 weeks, 6 weeks, 9 weeks and 15 weeks old mice, n = 4, 4, 6, 4, respectively, mean ± s.d. No significant difference by one-way ANOVA. C UMAP plots from single-cell sequencing, each dot represents one cell, the color represents the expression levels of Cx3cr1. D Histogram plot of Cx3cr1. The x-axis represents Cx3cr1 expression read counts; the y-axis represents cell numbers. Figure S4. Additional data related to Fig. 2. A Graph showing relative Iba-1+ microglia at 1 day, 3 days and 7 days after the final Tam injection, respectively; n = 3–4, mean ± s.d. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 by Student’s two-tailed unpaired t test. B Representative images of Iba-1 microglia staining of Cx3cr1CreER-Eyfp/wt mice and Cx3cr1CreER-Eyfp/wt Rosa26DTA/wt mice at 1 day after the final Tam injection in cortex and cerebellum. Scale bar, 200 μm. C Representative 3 sequential sagittal sections (25 µm/section, with 36 sections interval) after Hoechst staining of Cx3cr1CreER-Eyfp/wt Rosa26DTA/wt mice brains. The numbers represent the location and number of Cx3cr1highCre−Eyfp− microglia at 1 day and 3 days after the final Tam injection, each color represents one mouse, n = 3. D Quantitative data showing the total number of microglia in the 3 sections in Ctrl group, 1 day and 3 days after the final Tam injection, n = 4, 3, 3, mean ± s.d. Figure S5. Additional data related to Fig. 4. A–C Bar graphs showing RPKM of Cre, EYFP, and Cx3cr1, respectively, n = 5, mean ± s.d. ***p &lt; 0.001 by Student’s two-tailed unpaired t test. D The expression of CX3CR1 in both Cx3cr1CreER-Eyfp/wtCre+Eyfp+ and Cx3cr1highCre−Eyfp− microglia in Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice at day 42 after the final Tam injection. E Bar graph showing the mean fluorescence intensity (MFI) of CX3CR1, n = 4, mean ± s.d. ***p &lt; 0.001 by Student’s two-tailed unpaired t test. Figure S6. Additional data related to Fig. 4. A Schematic diagram showed how the primers were designed. The red color indicates the common primers used in all the PCR reactions; the green and blue color indicate the WT primer and Mutant primer used in PCR reaction for Fig. 4f; the orange color indicates the long PCR product targeting primer used in PCR reaction for Fig. 4g. B Whole-genome sequencing of Cx3cr1highCre−Eyfp− microglia in mouse 2. The top panel is the sequencing mapping on the CreERT2-Eyfp vector; the middle panel is the sequencing mapping on chromosome 9; the bottom panel is the schematic graph showing the homozygosity of the exon 2 of Cx3cr1. C Whole-genome sequencing of Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia. The top panel is the sequencing mapping CreERT2-Eyfp vector; the middle panel is the sequencing mapping the chromosome 9; the bottom panel is the schematic graph showing the heterogeneity of the exon 2 of Cx3cr1. D DNA electrophoresis of PCR product from EYFP− and EYFP+ microglia. E Representative dot plots showing the percentage of EYFP+F4/80low, EYFP−F4/80low, EYFP+F4/80high, and EYFP−F4/80high repopulated microglia at day 42 after Tam in Cx3cr1CreER-Eyfp/ CreER-Eyfp Rosa26DTA/wt mice, mean ± s.d.