Clonal and molecular changes in hematopoietic system upon acute platelet depletion revealed using RNA barcoding studies

ABSTRACT: Purpose: RNA cellular barcoding has enabled the analysis of anucleate platelets and their upstream progenitors and stem cells using single cell RNA seq, using Smart Seq2 Methods: Single stem cell, megakaryocytic progenitor or erythroid progenitors from transplanted animals with barcoded stem cells were used for Smart Seq2 protocol and sequenced on NovaSeq. Obtained from the sequencer Fastq files were used for alignment to Mus musculus transcriptome version M38 using STAR aligner and count reads (Dobin et al., 2013). Results: Subsequent analysis was performed in R using Seurat version 4.0.0(Papalexi et al., 2021). Cells showing gene counts lower than 50000 reads and a mitochondrial gene expression percentage higher than 10% were excluded from further analysis. Within Seurat, data were normalised using the NormalizeData function (using the LogNormalize method and scale.factor = 10000). We recovered 2569 single cells meeting above criteria from 8 analysed animals in total (4 control and 4 platelet depleted). Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE188268 | GEO | 2021/11/07

REPOSITORIES: GEO