*4.7. Quantitative Proteomic Analysis*

Proteomic analysis was performed at the Proteomics Core Facility at Sahlgrenska Academy, University of Gothenburg. For this purpose, the in vitro di fferentiated erythroid colonies were collected from the colony formation assay. The colonies were rinsed in PBS twice to remove any residues from the culture. The cell pellets were stored at −70 ◦C prior to lysis and protein extraction. The sample preparation and liquid chromatography-mass spectrometry process were carried out as explained in the Supplementary Materials and Methods [98]. Data analysis was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific, Stockholm, Sweden) against the Human Swissprot Database version March 2017 (Swiss Institute of Bioinformatics, Switzerland). Mascot 2.5 (Matrix Science, London, UK) was used as a search engine with precursor mass tolerance of 5 ppm and fragment mass tolerance of 200 mmu. Tryptic peptides were accepted with zero missed cleavage and variable modifications of methionine oxidation, cysteine alkylation and fixed modifications of N-terminal TMT-label and lysine TMT-label were selected. The detected peptide threshold in the software was set to false discovery rate (FDR) ≤0.01 by searching against a reversed database. Identified proteins were grouped by sharing the same sequences to minimize redundancy. Reporter ion intensities were quantified in MS2 spectra at Minimum Quan Value Threshold set to 2000. The resulting ratios were normalized in the Proteome Discoverer 1.4 on the median protein value of 1.0 in each sample. Heat maps were generated using XLSTAT software.

#### *4.8. Fluorescent-Activated Sorting of Erythroblasts from the UCB*

To block Fc receptors, the isolated MNCs were incubated with FcR Blocking Reagent (Miltenyi Biotec) at 4 ◦C for 30 min. The cells were rinsed and pelleted 300 × *g* at 4 ◦C. After resuspension and filtering through a 50 μm cup-shaped filter (BD Biosciences), the cells were stained for di fferent surface markers using the following mouse anti-human antibody (ab)-conjugation set: CD45-FITC [1:25], CD235a/Glycophorin A (GPA)-BV421 [1:100] (BD Biosciences), CD49d/Integrin alpha 4- phycoerythrin/Cy7 [1:300] (BioLegend, Täby, Sweden) and CD233/Band 3-phycoerythrin [1:300] (Bristol Institute for Transfusion Sciences). After 30 min incubation on ice, the cells were rinsed and analyzed by a BD FACSAria ™ I. 7AAD (Sigma Aldrich, Stockholm, Sweden) at a final concentration of

10 μg/mL was used as a viability marker. Spectral compensation was carried out using VersaComp Antibody Capture Beads kit (Beckman Coulter) and the gates were set based on unstained and fluorescent minus one controls. The data analysis was performed using FlowJo (version 10.0.8).

Considering the few numbers of the early-stage erythroid precursors, all the cells from proerythroblasts (CD45− GPA+ CD49dhi Band 3−) to reticulocytes (CD45− GPA+ CD49dlo Band 3<sup>+</sup>) were pooled together for each arterial and venous UCB sample. A total of 10<sup>6</sup> cells were sorted and collected for each sample. The cells were centrifuged at 300× *g* for 10 min, lysed in 350 μL of Buffer RLT from AllPrep DNA/RNA/Protein Mini Kit (Qiagen) according to the manufacturer's protocol and stored at −80 ◦C until later processing.

#### *4.9. RNA Extraction, Library Preparation and Quality Check*

All samples were thawed on ice and processed for RNA extraction using AllPrep DNA/RNA/Protein Mini (Qiagen) according to manufacturer's instructions. All centrifugations were performed at RT at 9000× *g* and the RNA was eluted in 50 μL RNase-free water. RNA samples were frozen at −80 ◦C and thawed for quality check and library preparation. RNA integrity was analyzed using Agilent RNA 600 Nano Kit (Agilent Technologies, Santa Clara, CA, US) on a Bioanalyzer 2100 (AgilentTM, Santa Clara, CA, US) according to the manufacturer's instructions. All the samples indicated an RNA integrity number (RIN) ≥ 9 and were used for library preparation and amplification by the QuantSeq 3 mRNA kit (LexogenTM, Vienna, Austria) according to the manufacturer's protocol. Quality and sequence length of the libraries were assessed by Fragment analyzer using the High sensitivity NGS fragment analysis kit (Advanced Analytical Technologies, Inc., Heidelberg, Germany), while concentration evaluation was performed by BioTek™ Synergy™ 2 (BioTek Instruments, Inc., Winooski, VT, US) microplate reader using Quant-iTTM PicoGreenTM dsDNA Assay Kit (InvitrogenTM, Carlsbad, CA, US) for a low-range assay. The RNA library was sequenced in SE50bp mode on an Illumina HiSeq 2500 (Illumina, San Diego, CA, US).
