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Single-cell RNA sequencing facilitates quantitative analysis of mouse hematopoietic stem/progenitor cells and SCF/2i expanded cells

ABSTRACT: Single-cell RNA sequencing facilitates quantitative analysis of mouse hematopoietic stem/progenitor cells and SCF/2i expanded cells

PROVIDER: PRJNA631793 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11773500_1.fastq.gz	Fastqsanger.gz
	SRR11773500_2.fastq.gz	Fastqsanger.gz
	SRR11773501_1.fastq.gz	Fastqsanger.gz
	SRR11773501_2.fastq.gz	Fastqsanger.gz
	SRR11773502_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA sequencing facilitates quantitative analysis of mouse hematopoietic stem/progenitor cells and SCF/2i expanded cells

Project description:Purpose: The goal of this study is to compare the transcriptome profiling of SCF/2i expanded cells to freshly sorted hemtopoietic stem/progenitor cells (HSPCs) at single cell level. Methods: Mouse bone marrow HSPCs (HSC, MEP, CLP, CMP, GMP) and alive SCF/2i expanded cells were sorted by FACS. The FACS-sorted single cell suspensions were washed with ice cold 0.04% BSA in PBS, then loaded onto 3’ library chips as per the manufacturer’s protocol for the Chromium Single Cell 3’ Library (10X Genomics, V2). The 10× Genomics libraries were first sequenced on an Illumina NextSeq 500 aiming at a coverage of 5,000 raw reads per cell to estimate the cell numbers, before being sequenced deeper on an Illumina HiSeq 2500 aiming at a coverage of 50,000 raw reads per cell. (paired-end; read1: 26 cycles; i7 index: 8 cycles; read 2: 98 cycles). Raw sequencing data were pre-processed using the Cell Ranger pipeline (10× Genomics, v 2.1.0). The raw-count datasets from difference cell types were integrated and analyzed by Seurat3 R package. Results: tSNE plot showed that the transcriptome of SCF/2i cells overlapped with that of freshly sorted GMPs. The top 50 highly expressed genes from each comparison were used for analysis by heatmap, which showed that the SCF/2i cells shared a similar gene expression pattern with GMPs, but not other hematopoietic cell lineages. Conclusion: The SCF/2i expanded cells are GMPs.

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Scf-GFP+ cells from the bone marrow and whole bone marrow microarray

Project description:The HSC niche factor SCF is required for HSC maintenance. Using an Scf-GFP knockin mouse, we have identified a perivascular cell type in the bone marrow expressing high level of Scf. To characterize the novel Scf-GFP+ cells from the bone marrow, we performed microarray analysis on these cells. Total RNA were isolated from 3 independent, freshly aliquots of FACS sorted 5,000 SCF-GFP+ cells or whole bone marrow cells isolated from young adult mice. Purified RNA was amplified using the WT-OvationM-bM-^DM-" Pico RNA Amplification system (NuGEN Technologies). Sense strand cDNA was generated using WT-OvationM-bM-^DM-" Exon Module (NuGEN), then fragmented and labeled using the FL-OvationM-bM-^DM-" cDNA Biotin Module V2 (NuGEN). 2.5M-BM-5g of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.

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Project description:We report the application of genome-wide RNA-sequencing analysis for pluripotent mouse ES cells maintained in 2iL or 2i and differentiated for 12, 24, 36, 48, 60, 72, 84, 96 hours. We find that cells differentiating from both 2iL and 2i follow a similar trajectory with 2i samples accelerated by 24 hours. Moreover we performed unsupervised hierarchical clustering on cells differentiating from 2iL and defined 6 groups of genes (gene signatures) based on their transcriptional behaviour during differentiation. Finally we reported that only after activation and dissolution of the formative signatures cells are fully committed. The same behaviour is true for cells differentiating from 2i.

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