Additional file 2 of Myelodysplastic syndromes are multiclonal diseases derived from hematopoietic stem and progenitor cells
Creators: name: Luo, Bingqing
name: Dong, Fang
name: Qin, Tiejun
name: Zhang, Qingyun
name: Bai, Haitao
name: Wang, Jinhong
name: Jia, Yujiao
name: Ma, Shihui
name: Jiang, Erlie
name: Cheng, Tao
name: Xiao, Zhijian
name: Ema, Hideo
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 2: Figure S1. Gating strategy for seven cell populations. FACS strategy used to isolate single cells from the seven populations: H, CD34+CD45low cells; E, CD45−CD235a+CD71+ cells; M, CD45−CD235a−CD41+ cells; m, CD45+CD33+CD14+CD16− cells; n, CD45+CD33+CD14−CD16+ cells; B, CD45+CD19+CD20+ cells; and T, CD45+CD3+ cells. The upper panels represent HD data, and the lower panels represent data from a patient with MDS. Figure S2. Targeted single-cell RNA-seq method. A Schematic diagram of the targeted single-cell RNA-seq method. Single H, n, m, E, M, B, and T were sorted by flow cytometry into a 96-well plate. One-step RT-PCR was performed with the first-round primer mix. The second round of PCR was performed with PCR primer mix 1 or mix 2. Finally, a sequencing library was constructed with p5 and p7 adaptors. B Frequency at which the DNMT3A p.R301W mutation was detected in 32 single JURKAT cells. The positive rate was 78.1%. C Electrophoretogram of the representative libraries from single cells and 20 cells from a patient with MDS. One pair of each primer (U2AF1, SF3B1, ASXL1, TP53, TET2, and DNMT3A) from the PCR primer mix (PCR primer mix 1 or PCR primer mix 2) was used for the second round of PCR, followed by library construction. PCR products were separated on a gel. Library construction performed without cells served as a negative control (neg). Figure S3. Qualification of single cell RNA-seq data. A The results of single-cell filtration. The number of qualified and unqualified single cells is shown for individual donors. Only qualified cell data were used in the analysis. B Coverage of each mutation in single cells after filtration. The total number of reads covering each mutation site in qualified single cells is shown as the coverage. The threshold of coverage was 10x and is marked by a horizontal dotted line. Data are presented as the medians with interquartile ranges. Data points that fell outside of the upper and lower whiskers were considered outliers and are not shown. C Variant allele frequency (VAF) of mutations after filtration. Each dot represents the VAF of each mutation from single cells of donors carrying this mutation after filtration. Figure S4. The clone landscape in all donors. A Data include all clones identified. Eighteen identified mutations were used to detect eighty-nine clones from all donors. Each column represents a clone. CH clones were identified with CH mutations. MDS clones were identified with MDS mutations regardless of the coexistence of CH mutations. The lineage distributions of clones are shown in orange in the upper panel. The composition of mutations in each clone is shown in the lower panel. The presence of mutations is shown in red. WT is shown as blank. Mutation sites without enough reads to determine genotypes are shown in gray as not available (NA). The bottom panel shows the clone size. Wild type (WT) clones without mutations were excluded. Data of HD3 and HD5 are not shown since neither mutations nor clones were detected. B Clone size of CH clones and MDS clones from all donors. ***, p &lt; 0.001 (Mann–Whitney U test). C Clone size and number of populations involved in clones were compared between low blasts and high blasts groups. Fourteen patients with MDS were classified into low blasts (1%-9%) group (n = 8) mainly consisting of EB1 and high blasts (&gt;9%) group (n = 6) mainly consisting of EB2. ns, p &gt; 0.05 (Mann–Whitney U test).