Single cell lipidome analysis of phosphatidylcholines and spingomyelins from prostate cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): Cultured Cells
SUBMITTER: 
Sarah Hancock
PROVIDER: ST002367 | MetabolomicsWorkbench | Sun Oct 30 00:00:00 BST 2022
REPOSITORIES: MetabolomicsWorkbench
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2013-09-09 | GSE50690 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS. We compared RNA quantitation between unfixed and formaldehyde-fixed cells using the NanoString nCounter system. This analysis was performed using two cell lines, K562 and CEMx174, and human PBMCs. Three biological replicates were performed for each cell type. We also performed this comparison using human PBMCs sorted by FACS. For this analysis, we isolated PBMCs from two donors and performed two technical replicates for each donor sample.
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Project description:BLaER1 cells are human leukemia pre-B cells able to transdifferentiate into functional and non-tumorigenic macrophages. With the goal of uncovering the genes involved in the transdifferentiation process, we have designed a DECKO (Double Excision CRISPR Knockout) library to knockout lncRNAs and protein coding genes (pc-genes) overexpressed along the seven days the process lasts. We have seen that targeting pc-genes with two gRNAs synergistically enhances the efficiency of knock out, by inducing deletions and/or frameshifts that promote the expression of non-functional proteins. Thus, using the CRISPETa tool, we designed paired guide RNAs targeting either the region surrounding the Transcription Start Site of the 166 lncRNAs or the coding exons of the 874 pc candidate genes. Cas9-expressing BLaER1 cells were infected at low-multiplicity of infection with the combined library and induced transdifferentiation. Delayed and differentiated subpopulations of cells were isolated by Fluorescence-Activated Cell Sorting at 3 days and 6 days after induction, and pgRNAs were sequenced in an Illumina HiSeq2500 instrument. The sequencing reads were mapped and quantified to uncover the enriched pgRNAs found in each subpopulation. Among all genes targeted in the library, we identified twenty pc-genes and six lncRNAs as strong candidates to be involved in the process, as the pgRNAs targeting their loci were enriched in the delayed population compared to the differentiated one, either at 3 days or at 6 days after transdifferentiation induction. From these, we selected two pc-genes and two lncRNAs for deeper characterization.
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