DOI: 10.5061/dryad.573n5tb77
title: The transcriptomic landscape of normal and ineffective erythropoiesis at single cell resolution

description: Ineffective erythropoiesis, the death of maturing erythroid cells, is a
 common cause of anemia. To better understand why this occurs, we studied
 the fates and adaptations of single erythroid marrow cells from
 individuals with Diamond Blackfan anemia (DBA), del(5q) myelodysplastic
 syndrome (del(5q) MDS), and normal controls, and defined an unhealthy (vs.
 healthy) differentiation trajectory, using velocity pseudotime and cell
 surface protein assessment. The pseudotime trajectories diverge
 immediately after the cells upregulate transferrin receptor (CD71), import
 iron, and initiate heme synthesis, although cell death occurs much later.
 Cells destined to die highly express heme-responsive genes, including
 ribosomal protein and globin genes. In contrast, surviving cells
 downregulate heme synthesis, while upregulating DNA damage response,
 hypoxia, and HIF1 pathways. Surprisingly, 24±12% of cells from controls
 follow the unhealthy trajectory, implying that heme also regulates cell
 fate decisions during normal red cell production. Del(5q) MDS (unlike DBA)
 results from somatic mutations, so many normal (unmutated) erythroid cells
 persist. By independently tracking their trajectory, we gained insight
 into why they cannot expand to prevent anemia. In addition, we show that
 intron retention is especially prominent during red cell differentiation.
 The additional information provided by messages with retained introns also
 allowed us to align data from multiple independent experiments and thus
 accurately query the transcriptomic changes that occur as single erythroid
 cells mature.
description: Cryo-preserved bone marrow mononuclear cells were thawed,
 resuspended in equivalent volume of FBS, washed in HBSS, then resuspended
 at 1×10<sup>7</sup> cells per ml in PBS, 0.1% BSA, and 2 mM
 EDTA, and incubated with 1:10 dilution of biotinylated anti-CD3,
 anti-CD19, and anti-CD11b. Antibody-bound lineage-positive cells were
 depleted with streptavidin beads. Lineage-depleted marrow mononuclear
 cells were cultured in erythroid expansion media (IMDM, 5% pooled Human AB
 plasma, 330 μg/ml holo-transferrin, 1 μM dexamethasone, 160 mM
 monothioglycerol, 10 μg/ml Insulin, 2 U/ml preservative-free heparin, 2
 U/ml EPO, 100 ng/ml SCF and 5 ng/ml IL3) at 2×10<sup>5</sup>
 cells/ml. The cells were re-seeded at 2E5 cells/ml on day 3 with fresh
 media. Cells were collected on days 0, 3, and 6.  Cells
 were washed and resuspended in PBS with 1% BSA at
 5×10<sup>5</sup> per ml.  Cells were incubated with 10 μg/ml
 of each DNA-labelled antibody (Biolegend TotaSeq or equivalent): CD3,
 CD11b, CD14, CD36, CD70, CD71/TfR, CD117/cKit, CD235a/GlyA.  Sample
 processing. Single-cell mRNA barcoding and library preparation were
 performed on the 10x Chromium controller according to the manufacturer’s
 instructions. A single GEM (gel beads-in-emulsion) well from each sample
 was used to generate both the gene expression and antibody capture
 libraries which were recombined for sequencing. The libraries were
 sequenced on Illumina NextSeq 550 or NovaSeq instruments and raw sequence
 data was processed with Cell Ranger (version 3). Bam
 files produced by Cell Ranger were analyzed by Velocyto and transcript
 splice quantification saved as Loom files. Loom files were aggregated by
 ScanPy (concatenate function). Transcript counts were normalized to
 1×10<sup>4</sup> UMIs per cell and log-transformed (log1p
 function). The aggregated data was saved to a single HDF5 file.
description: Scanpy (https://scanpy.readthedocs.io/en/stable/)