Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. Young hematopoietic stem cells (HSCs) were isolated by flow cytometry, transduced ex vivo with different shRNA constructs (shRen, shPU1 No1, shPU1 No2) and transplanted into lethally irradiated mice. Two months after transplantation, transduced (GFP+)HSPCs were isolated by flow cytometry. The different samples were pooled and the shRNA per cell was identified by a specific dialout PCR (amplifying the mirE shRNA construct together with the 10x cell barcode) to link the cell barcodes with the the different shRNA constructs. Here mirE_R1 contains the UMI and the cell barcode whereas mirE_R2 contains the shRNA sequence.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. HSPCs were sorted by flow cytometry. During the staining procedure for flow cytometry, the antibody cocktail also included antibodies labelled with oligos (antibody-derived tags =ADTs) which can later be analyzed by the 10x Chromium controller.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. HSPCs were sorted by flow cytometry. LT-HSCs were fractionated into a Clca3a1 high or low population using a monoclonal antibody (10.1.1) purchased from the Developmental Studies Hybridoma Bank - DSHB.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. HSPCs were sorted by flow cytometry. Where indicated, LT-HSCs were further fractionated into a Clca3a1 high or low population using a monoclonal antibody (10.1.1) purchased from the Developmental Studies Hybridoma Bank - DSHB.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. HSPCs were sorted by flow cytometry. Where indicated, LT-HSCs were further fractionated into a Clca3a1 high or low population using a monoclonal antibody (10.1.1) purchased from the Developmental Studies Hybridoma Bank - DSHB. We report here the analysis of old LSK cells after Taz S89A overexpression. LSK cells were sorted by flow cytometry. The cells were infected in vitro with the indicated constructs and subjected to RNA-Seq analysis. We report here the analysis of BM-HPC#5 cells after PU1 knockdown. BM-HPC#5 cells with the indicated constructs were subjected to RNA-Seq analysis.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. Old (Clca3a1_high) hematopoietic stem cells (HSCs) were isolated by flow cytometry, transduced ex vivo with different shRNA constructs (shRen, shTaz_Seq1, shTaz_Seq2) and transplanted into lethally irradiated mice. Four months after transplantation, transduced (GFP+)HSPCs were isolated by flow cytometry and subsequently analyzed by low input RNA-Seq. Per sample two mice were pooled in order to obtain sufficient RNA for library prep.
