Figure 2.

RNA-Seq analysis and SDE gene identification from blood Ly6C^high and Ly6C^low MC of C57/BL6 control and Cbs ^-/- mice. (A) Cbs ^-/- mice and Hcy levels. Eleven mice were used in each group. Severe HHcy were determined in Cbs ^-/- mice (plasma Hcy 128.13 μmol/L). (B) Gating and sorting strategy to isolate Ly6C^high and Ly6C^low MC. Mouse white blood cell were prepared from peripheral blood and stained with antibody against CD11b, Ly6G and Ly6C and subjected for flow cytometry cell sorting. Intact cells (72.8%) were recognized based on higher FSC-A (larger size). Singlets (71.0%) were identified by using FSC-H versus FSC-A appeared on a diagonal. CD11b⁺Ly6G^- cells (11.0%) were selected as MC. MC subsets (CD11b⁺Ly6G^-Ly6C^high, and CD11b⁺Ly6G^-Ly6C^low) were sorted based on Ly6C levels. The quantification of MC was used flow cytometry analysis for Ly6C^high and Ly6C^low MC in CT and Cbs^-/-. (C) General data. 100 ng mRNA were obtained from 100,000 sorted cells and achieved around 30 million reads and 16,487 normalized genes per sample by mRNA-Seq analysis. (D) PCA plot. PCA analysis incorporated 8 samples from 4 groups of MC subsets {Ly6C^high (CT), Ly6C^low (CT), Ly6C^high (Cbs^-/-) and Ly6C^low (Cbs^-/-), n=2} using the R software package Seurat. PC1 versus PC2 demonstrates the close transcriptional proximity. PC1, PC2 and PC3 variance is 44.1%, 21.1% and 12.9%. PC1 (44.1%) means that the difference on the x-axis can explain 44.1% of the overall result. (E) Comparison strategy and SDE gene identification. We performed four group comparison (A–D) and identified down-regulated and up-regulated SDE genes using the criteria of |Log₂FC| more than 1 (2-FC) and adjusted P value less than 0.01. (F) SDE genes in four comparison groups. Volcano plot of all genes demonstrates the expression pattern of SDE genes in four comparison groups. Down-regulated SDE genes are highlighted in green and up-regulated in red (|Log₂FC|>1, adj. P<0.01), with Log₂FC as x-axis and −Log₁₀(adjust P-value) as y-axis. MC, monocyte; CT, control; Cbs, cystathionine β-synthase; HHcy, Hyperhomocysteinemia; Hcy, homocysteine; FACS, fluorescent-activated cell sorting; PCA, principal component analysis; PC, principal component; SDE, significantly differentially expressed; FC, fold change.