Immune Transcriptome Study of Human Nucleated Erythroid Cells from Different Tissues by Single-Cell RNA-Sequencing

Purpose: studying of immunity-related genes in CD235a erythroid cell in order to validate previous findings on the subject using single cell RNA sequencing (scRNA-seq) and its benefits such as mRNA co-localization in a single cell and cell specific marker gating of populaions of interest. Methods: we extracted mononuclear cells from the fetal liver, the fetal thymus, the cord blood and the adult bone marrow, performed magnetic separation of CD235a (Erythroid) cells, marked magnetically separated cells with BD Sample Tags, loaded the cells on a BD Rhapsody cartridge, lysed the cells, performed reverse transcription, cDNA library preparation, QC of final cDNA libraries by BioAnalyzer 2000 and Qubit 4, pooled the final cDNA libraries, sequenced them on a Illumina NextSeq 550 sequencer. Then, we processed FASTQ R1 and R2 files using the BD Pipeline V1.9 and got final metrics in a mols_per_cell.tsv file. Results: in this work, we investigated other possible immunity-related functions on a single cell level using RNA sequencing of adult bone marrow, cord blood and fetal liver NECs and found that NECs from every studied origin expressed ARG1, CXCL5, CXCL8, DEFA3, DEFA4, IL1B, IL15, IL18, IGHE (secreted), LGALS3, LAGLS9, TGFB3 and VEGFA. Conclusions: our study allows to access new potential immunological roles of CD235a erythroid cells, both validated and discarded previous results regarding cytokine and chemokine gene expression in erythroid cells. Single cell immunity-related gene profiles of CD235a Erythroid cells from the adult bone marrow, from the cord blood, from the fetal thymus and from the fetal liver. Sample Tag 1-3 = Cord Blood, n=3, 1620 cells were called in total (Sample_ID 1 in the dataset). Sample Tag 3-6 = Fetal liver parenchyma, n=3, week 20-22 of development, 3690 cells were called in total (Sample_ID 2 in the dataset). Sample Tag 7-9 = Fetal thymus parenchyma, n=3, week 20-22 of development, this sample was discarded (only 37 single cells were called in total). Sample Tag 10-12 = Adult bone marrow, n=3, 24-26 years old males, 3799 cells were called in total (Sample_ID 4 in the dataset).