DOI: 10.5281/zenodo.5645044
title: Erythropoietin directly remodels the clonal composition of murine hematopoietic multipotent progenitor cells

description: ## In version 1 of the repository the scRNAseq data was corrupted -- in version 2 this has been corrected and the bam files of both scRNAseq runs have been uploaded ##

This dataset consists of the raw sequencing files of 9 independent barcoding experiments and of one 10X Genomics scRNAseq experiment. These are the source files for the main figures of the associated publication.

HSPCs (C-Kit+ Sca1+ CD150+ Flt3-) or MPP2 (C-Kit+ Sca1+ Flt3- CD150+ CD48+) were isolated form mouse bone marrow through flushing, MACS-enrichment and sorting. The cells were barcoded by spin infection for 6h, and cultured in StemSpanMedium SFEM with 50 ng/ml mSCF (STEMCELL Technologies) for 16h with or without human recombinant EPO (Eprex, erythropoietin alpha, Janssen) at 1000 ng/ml or 160 ng/ml. At this stage scRNAseq was perfromed  on the 10X Chromium platfom (10X Genomics), or cells were transplanted by tail vein injection into 6Gy irradiated recipient mice. When appropirate an additional injection of EPO 133ug/kg was given at the moment of transplantation. Different mature hematopoietic cells were isolated form the transplanted mice after 4 weeks or 16 weeks.

For barcoding experiments, a three-step PCR was performed to amplify barcode sequences, to add Read1 and Read2 Illumina sequencing adapters, P5 and P3 flow cell attachment sites  as well as plate and sample indices. Libraries were sequenced on an Illumina HiSeq SR65 with 10% of PhiX spike-in. For scRNAseq experiment, libraries were made using the Chromium SIngle Cell 3' v2 kit and sequencing was performed on a HiSeq PE26-98.

The repository encompasses the following datasets:



A1006.tar.gz -- AE05Low -- HSPCs EPO 160 ng/ml + injection 4 weeks -- main figure 2

A1007.tar.gz -- AE05High -- HSPCs EPO 1000 ng/ml + injection 4 weeks -- main figure 2

A984.tar.gz -- AE03Low -- HSPCs EPO 160 ng/ml 4 weeks -- main figure 2 and HSPC part of main figure 4

A1008.tar.gz -- AE03High -- HSPCs EPO 1000 ng/ml 4 weeks -- main figure 1, 2 and DC part of main figure 3

A1012.tar.gz -- AE04Low -- HSPCs EPO 160 ng/ml 4 months -- main figure 8

A1013.tar.gz -- AE04High -- HSPCs EPO 1000 ng/ml 4 months -- main figure 8

A1105.tar.gz -- AE07part1 -- HSPCs EPO 1000 ng/ml 4 weeks part 1 -- MkP part of main figure 3

A1107.tar.gz -- AE07part2 -- HSPCs EPO 1000 ng/ml 4 weeks part 2 -- MkP part of main figure 3

A1166.tar.gz -- AE13part1 -- HSPCs EPO 1000 ng/ml 4 weeks part 1 -- HSPC part of main figure 4

A1166.tar.gz -- AE13part2 -- HSPCs EPO 1000 ng/ml 4 weeks part 2 -- HSPC part of main figure 4

D757_3697.tar.gz -- LP26MPP2 -- MPP2 EPO 1000 ng/ml 4 weeks -- main figure 7


In version two of the repository the scRNAseq data is changed into:



possorted_genome_bam_T1.bam -- 10X of EPO exposed (T1) HSPCs -- main figure 5 and 6

possorted_genome_bam_T2.bam -- 10X of control (T2) HSPCs -- main figure 5 and 6


The folder for each barcode experiment encompasses fastq files for each sample index used in the experiment. These have to be further de-multiplexed by plate index and barcode reads have to be called. These, and all subsequent processing steps till the making of the main figures are described in the github folder accompanying the associated publication.