Project description:the aim 1 was to evaluate hashing (sample multiplexing) accuracy of TotalSeq-B antibodies and CellPlex (a multiplexing solution from 10x Genomics) on 2 pools of PBMCs from 3 different donors. Thus the genetic difference between donors was used as a ground truth for sample demultiplexing based on antibody or lipid hashing hashing. the aim 2 was to evaluate TotalSeq antibody and lipid hashing on different mice primary tissues using different hashing protocols.
Project description:Although classical single cell RNA analysis is a very powerful tool, it nevertheless has several drawbacks. To overcome these two disadvantages, the cell hashing technique seems interesting. In this work, we propose to test the feasibility and the reliability of the single cell hashing technique on primary AML cells. For this purpose, we compared the transcriptomic profile of AML cells analyzed by the classical single-cell RNA sequencing approach versus the cell hashing technique.
Project description:Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with and without TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation maintained experimental flexibility while similarly down-regulating ASG expression compared to TTis. Notably, addition of TTis during cold isolation offered minimal additional advantages. Thus, while both TTis and cold isolation effectively reduced ASG expression, we found cold isolation to be a more practical approach.
Project description:We performed single-cell RNA-seq analysis with CITE-seq and cell hashing of total viable aortic cells from Ldlr-/- mice fed a high fat diet for 13 weeks