Unknown,Transcriptomics,Genomics,Proteomics

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Fluidigm C1 + Illumina HiSeq quantitative whole transcriptome analysis of unsorted population of E16.5 lung cells

ABSTRACT: We used microfluidic single cell RNA-seq on mixed e16.5 mouse lung cells in order to determine the potential cell types present based on differential transcriptional profiles of the entire population using minimal cell selection bias. whole lung mouse e16.5 cells were pooled and loaded onto the Fluidigm C1 device. microwells that contained intact single cells were recorded, the labchip was processed for the generation of cDNA from each cell, and cDNA generated from each accepted well was used to generate amplified and barcoded DNA library that was loaded into an Iluumina HiSeq machine for HTS analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Yina Du

PROVIDER: E-GEOD-69761 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We used microfluidic single cell RNA-seq on 198 individual mouse lung epithelial cells at 4 different stages throughout development to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We classified 80 cells comprising the distal lung epithelium at E18.5 into distinct populations using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell types. This M-bM-^@M-^\reverse tissue engineeringM-bM-^@M-^] approach confirmed the basic outlines of the conventional model of cell type diversity in the distal lung and led to the discovery of a large number of novel transcriptional regulators and cell type markers that discriminate between the different populations. Moreover, we reconstructed the steps during maturation of bipotential progenitors into both alveolar lineages based on the presence of undifferentiated, differentiated as well as differentiation intermediate cells at the single time point E18.5. Finally, we followed Sftpc-positive cells throughout their lifecycle (E14.5, E16.5, E18.5, adult) and identified 7 gene sets that differentiate between the multipotential, bipotential, mature, as well as intermediate states of the AT2 lineage. 198 single-cell transcriptomes from mouse lung epithelium were analyzed in total, two 200-cell bulk control samples as well as one no-cell control; All single cell and control samples contain 92 external RNA spike-ins; For time point E18.5, three individual experiments were performed using 3 different pregnant mice (3 biological replicates): Replicate 1 (pooled sibling lungs) yielded 20 single cell transcriptomes, replicate 2 (one single embryonic lung) yielded 34 single cell transcriptomes and replicate 3 (pooled siibling lungs) yielded 26 single cell transcriptomes; In addition, a 200-cell bulk control sample was prepared for E18.5 replicate 1 and E18.5 replicate 3 experiments; A no-cell control sample was generated for the E18.5 replicate 1 experiment; For time point E14.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 45 single cell transcriptomes; For time point E16.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 27 single cell transcriptomes; For the adult time point, one 107 day old mouse was used and transcriptomes of 46 single cells were obtained; All single cell samples were processed on the microfluidic platform, 200-cell-bulk and no-cell control samples were processed in microliter volumes in PCR tubes.

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Fluidigm C1 + Illumina HiSeq quantitative whole transcriptome analysis of unsorted population of E16.5 lung cells

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