media. On day 7 and 10, cells were harvested and replated in complete media at a density of 1x10⁵ cells/mL. On day 14, cells were harvested and processed for flow cytometry.

High-performance liquid chromatography (HPLC) to detect hemoglobin tetramers was performed as previously described.²⁰⁶

## Differentiation of hPSC-derived HLF+ HOXA+ hematopoietic progenitors into macrophages

Day 10 hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted, and seeded in StemPro-34 base media (Thermo Fisher) supplemented with the following factors:

* Day 0-5: SCF (50 ng/mL [Peprotech, 300-07]), TPO (10 ng/mL [Peprotech, 300-18]), IL-3 (50 ng/mL [Peprotech, 200-03]), FLT3L (50 ng/mL [Peprotech, 300-19]), M-CSF (50 ng/mL [Peprotech, 300-25]), and ITS-X (Thermo Fisher, 51500-056)]

* Day 6-10: FLT3L (50 ng/mL), M-CSF (50 ng/mL), GM-CSF (25 ng/mL [Peprotech, 300-03]), and ITS-X

* Day 10-17: M-CSF (100 ng/mL), GM-CSF (50 ng/mL), and ITS-X

At day 17, cells were dissociated using TrypLE (Thermo Fisher), stained with CD11b APC antibody (BioLegend, 101212) and CD68 PE Cy7 antibody (Thermo Fisher, 25-0689-42) in the presence of human Fc blocking reagent (Miltenyi Biotec, 130-059-901), and analyzed for flow cytometry.

## Differentiation of hPSC-derived HLF+ HOXA+ hematopoietic progenitors into T cells

T cell differentiation was performed using the StemSpan T Cell Generation Kit (STEMCELL Technologies, 09940) following the manufacturer's recommendations. In brief, day 10 hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted, and seeded in StemSpan Lymphoid Progenitor Expansion Medium at a density of 1-2x10⁴ cells/mL (1x10⁴ cells/mL for CD34+ cord blood HSPCs) in plates pre-coated with Lymphoid Differentiation Coating Material. On day 3 (referring to 3 days of T cell differentiation, after the HLF+ HOXA+ hematopoietic progenitor stage), cells were supplemented with an equal volume of StemSpan Lymphoid Progenitor Expansion Medium. On days 7 and 10, half medium changes were performed. On day 14, cells were harvested, counted, and reseeded in StemSpan T Cell Progenitor Maturation Medium at a density of 1x10⁵-1x10⁶ cells/mL in plates pre-coated with Lymphoid Differentiation Coating Material. On day 17, cells were supplemented with an equal volume of StemSpan T Cell Progenitor Maturation Medium. On days 21 and 24, half media changes were performed. On day 28, cells were collected, counted, and processed for flow cytometry.

For further maturation into CD8+ single-positive T cells, T cells from day 28 were harvested, counted, and reseeded in StemSpan T Cell Progenitor Maturation Medium supplemented with IL-15 (10 ng/mL [Peprotech, 200-15]) and 12.5µL/mL ImmunoCult Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, 10970) at a density of 1x10⁶ cells/mL in plates pre-coated with Lymphoid Differentiation Coating Material. On day 31, cells were supplemented with an equal volume of StemSpan T Cell Progenitor Maturation Medium supplemented with IL-15. On day 35, cells were collected, counted, and processed for flow cytometry.

## Differentiation of hPSC-derived HLF+ HOXA+ hematopoietic progenitors into NK cells

NK cell differentiation was performed using co-culture with OP9-DLL4-IRES-GFP (alternatively known as "OP9-DLL4") feeder cells.¹⁴⁹ Day 10 hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted and reseeded onto a confluent layer of OP9-DLL4-IRES-GFP feeder cells at a concentration of 1x10⁴-1x10⁵ cells/mL (1x10³ cells/mL for CD34+ cord blood HSPCs) in αMEM (Thermo Fisher, 12-571-063) supplemented with 20% FBS (R&D Systems, S11550), 1% penicillin/streptomycin (Thermo Fisher, 15-140-122), SCF (30 ng/mL [Peprotech, 300-07]), FLT3L (5 ng/mL [Peprotech, 300-19]), IL-7 (5 ng/mL [Peprotech, 200-07]), IL-15 (10 ng/mL [Peprotech, 200-15]), and for the first week of culture only, IL-3 (5 ng/mL [Peprotech, 200-03]). Cells were supplemented with an equal volume of media on days 3 and 10, and cells were collected and reseeded onto fresh OP9-DLL4-IRES-GFP feeders on day 7. On day 14, cells were collected, counted, and processed for flow cytometry.

## NK cell killing assay

On day -1, 10³ OP9-DLL4-IRES-GFP feeder cells were seeded per well of a 96-well plate in αMEM (Thermo Fisher, 12-571-063) supplemented with 20% FBS (R&D Systems, S11550) and 1% penicillin/streptomycin (Thermo Fisher, 15-140-122). On day 0, NK cells derived from human CD34+ cord blood HSPCs or hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted, and MACS enriched for CD56+ cells following the manufacturer's instructions. In brief, cells were stained with anti-human CD56 APC antibody (BioLegend, 318310), and then they were stained with anti-APC microbeads (Miltenyi, 130-090-855) before performing magnetic separation.

After MACS enrichment, 10⁴ CD56+ NK cells were seeded onto OP9-DLL4-IRES-GFP feeders per well of a 96-well plate in αMEM + 20% FBS + 1% penicillin/streptomycin. Cells were cultured in an Incucyte Live-Cell Analysis System and imaged every hour for 72 hours. The total GFP+ area was measured to quantify NK cell killing efficacy over time.

## Differentiation of hPSC-derived HLF+ HOXA+ hematopoietic progenitors into B cells

B cell differentiation was performed using co-culture with MS5 feeder cells (DSMZ). Day 10 hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted, and reseeded onto a confluent layer of MS5 feeder cells at a concentration of 1-5x10⁵ cells/mL (1x10³-1x10⁴ cells/mL for CD34+ cord blood HSPCs) in αMEM (Thermo Fisher, 12-571-063) + 10% FBS (R&D Systems, S11550) + 1% penicillin/streptomycin (Thermo Fisher, 15-140-122) + SCF (100 ng/mL [Peprotech, 300-07]) + G-CSF
(10 ng/mL [Peprotech, 300-23]). Cells were supplemented with equal volume of media on days 3, 10, and 17, and cells were collected and reseeded onto fresh MS5 feeders on days 7 and 14. On day 21, cells were collected, counted, and processed for flow cytometry.

**Culture of human cord blood hematopoietic stem and progenitor cells**

Human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs [StemExpress, CB3400.5C]) were thawed and cultured in StemSpan II medium (STEMCELL Technologies, 09655) supplemented with SCF (20 ng/mL [Peprotech, 300-07]), TPO (50 ng/mL [Peprotech, 300-18]), FLT3L (20 ng/mL [Peprotech, 300-19]), IL-6 (20 ng/mL [Peprotech, 200-06]), UM171 (75 nM [ApexBio, A8950]), and SR1 (750 nM [Cellagen Technology, C7710-5]). Cells were seeded at a concentration of 2x10⁵ cells/mL in U-bottom tissue culture treated plates. Each day, cells were gently triturated, and half of the cell suspension was transferred to a new well with all wells supplemented with fresh media to maintain the culture at low density. CD34+ cord blood HSPCs were either used immediately after thaw, or were cultured 3 days or less for all assays.

**Dissociation, antibody staining, and flow cytometry analysis of cultured cells**

Different methods were used to dissociate distinct types of cultured cells for flow cytometric analysis. First, undifferentiated and differentiated hPSCs were typically dissociated by incubation in TrypLE Express (Thermo Fisher, 12604013) for 3-5 minutes at 37 °C, followed by gentle trituration. Second, hPSC-derived hematopoietic progenitors were also more gently dissociated by incubation in Papain (0.5 mg/mL; Worthington Biochemical, LS003118) dissolved in PBS + 1.1 mM EDTA (Thermo Fisher, 15575020) + 0.067 mM 2-mercaptoethanol (Gibco, 21985023) + 5.5 mM Cysteine-HCl (Sigma, C7477) for 30-45 minutes at 37 °C, followed by gentle trituration, as described above. Third, hPSC-derived downstream blood and immune cells as well as cord blood HSPCs were less adherent and were therefore simply dissociated by gentle trituration.

After dissociation, cells were diluted 1:10 in DMEM/F12 and centrifuged at 500g for 5 minutes at 4 °C. Cell pellets were resuspended in FACS buffer (PBS + 1 mM EDTA [Thermo Fisher] + 2% v/v FBS [R&D Systems] + 1% penicillin/streptomycin [Thermo Fisher]). Generally speaking, fluorophore-conjugated primary antibodies were added to cells in FACS buffer and incubated for 30 minutes at 4 °C, protected from light. After staining, cells were washed twice with FACS buffer and resuspended in FACS buffer supplemented with either DAPI (1:10,000, Biolegend) or Propidium Iodide (PI; 1:1,000, Biolegend) for live/dead cell discrimination. Cells were passed through a 40 µM filter prior to flow cytometry. Flow cytometry was performed on a Beckman Coulter CytoFlex Analyzer, BD FACSymphony A5, or BD FACS Aria II SORP. All data analysis was done in FlowJo. Cells were first gated based on forward and side scatter, followed by height and width for doublet discrimination. Subsequently, live cells were identified as those that did not stain for DAPI or propidium iodide.

Flow cytometry compensation was performed using compensation beads (BD Biosciences, 552845 and 552843).

**Quantitative PCR**

RNA extraction, reverse transcription, and quantitative PCR (qPCR) was generally performed as described previously.¹⁰³ Cultured cells were directly lysed in the culture plate, or alternatively, were purified via FACS and then lysed, using 350 µL of RLT Plus Buffer. RNA was then extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's protocol. 300 ng of total RNA was reverse transcribed into cDNA for qPCR using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. qPCR was performed in 384-well format as previously described,¹⁰³ using gene-specific forward and reverse primers, as well as the SensiFAST SYBR Green Lo-ROX Kit (Thomas Scientific), on a QuantStudio 5 qPCR machine (Thermo Fisher). qPCR primer sequences are provided in Table S4. Expression of all genes was normalized to the levels of the reference gene YWHAZ.

**Immunocytochemistry of hPSC-derived cell types**

Adherent cells were washed with PBS, fixed in 4% paraformaldehyde for 10 minutes at room temperature, washed three times in PBS for 5 minutes each, and then permeabilized and blocked in blocking buffer (PBS + 0.1% Triton X-100 + 5% donkey serum) for 1 hour at 4 °C. Semi-adherent cells were harvested from the cell culture plate with TrypLE Express (Thermo Fisher, 12604013) and gentle trituration (as described above), then transferred to slides using a Cytospin cytocentrifuge (500g for 5 minutes), left to dry overnight, fixed in 2% paraformaldehyde for 10 minutes at room temperature, and then left to dry for 2 hours at room temperature.

Fixed cells were then incubated overnight in primary antibodies diluted in blocking buffer in a humidified chamber at 4 °C. The following day, cells were washed three times in PBS for 10 minutes each at room temperature and stained with Alexa Fluor-conjugated secondary antibodies diluted in blocking buffer for 1 hour at 4 °C. Cells were washed twice in PBS for 5 minutes each, and then stained with DAPI in PBS for an additional 5 minutes. Images were captured on an Olympus FV3000 confocal microscope. Images were rendered and analyzed with FIJI/ ImageJ software.

**Bulk-population RNA-sequencing of differentiated hPSCs and comparison with primary human hematopoietic stem and progenitor cells**

For bulk population RNA-sequencing, we employed fluorescence activated cell sorting (FACS) to purify (1) CD144+ CD45+ hPSC-derived day 10 hematopoietic progenitors, (2) CD34+ CD38- CD90+ CD45RA- Lineage- cord blood HSCs, (3) CD34+ CD38- CD90- Lineage- cord blood multipotent progenitors (MPPs), and (4) CD34+ CD38+ CD90- Lineage- cord blood downstream hematopoietic
progenitors (Figure S7C), following previously-published cell surface marker definitions of cord blood HSPCs.²⁰⁷,²⁰⁸ For FACS purification of cord blood HSPCs, we stained with the following antibodies — eFluor 450 Anti-human CD34 antibody (Thermo Fisher, 48-0349-42), FITC Anti-Human CD90 antibody (BD Biosciences, 555595), PE Anti-human CD45RA antibody (Biolegend, 304108), and APC Anti-Human CD38 antibody (BD Biosciences, 340439) — and also we employed a cocktail of PE Cy5-conjugated “lineage” antibodies to exclude differentiated blood and immune lineages, which recognized the following cell-surface markers: CD2, CD3, CD4, CD7, CD8, CD11b, CD14, CD16, CD19, CD20, CD56, and CD235A/GPA (BD Biosciences, 555328, 555341, 555348, 555362, 555368, 555389, 562335, 561725, 555414, 555624, 555517, 559944).

hPSC-derived cells or primary cord blood HSPCs were either directly lysed in the culture plate, or purified via FACS as indicated and then lysed, using 350 µL of RLT Plus Buffer and RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's protocol. RNA concentration and quality was assessed by Agilent Bioanalyzer using the High Sensitivity RNA Analysis kit (Agilent, 5067-1513), to ensure that RNA was of high quality (RNA integrity number [RIN] > 7).

1 ng of total RNA was further processed to synthesize cDNA using the SMART-Seq2 pipeline.²⁰⁹ cDNA size distribution and concentration were assessed by Agilent Bioanalyzer using the High Sensitivity DNA Analysis kit (Agilent, 5067-4626). The Illumina Nextera XT DNA Sample Preparation kit (Illumina, FC-131-1096) was used for library construction and the KAPA Library Quantification Kit (Kapa Biosystems, KK4854) was used for library quantification. Library size distribution was assessed by Agilent Bioanalyzer. RNA-sequencing libraries were sequenced on the Illumina NovaSeq 6000 sequencer, to obtain 2x150bp paired-end reads (PE150).

Kallisto¹⁵³ was used to align reads to the hg38 human reference genome (Homo_sapiens.GRCh38.96.gtf, downloaded from Ensemble) and to quantify transcript level abundances. Default parameters were used for quantification, and data were imported into DESeq2¹⁵¹ using tximport¹⁵⁵ for all downstream analysis in R, including differential gene expression analysis.

Bulk population RNA-sequencing data of FACS-purified CD144+ CD45+ HSCs and CD144- CD45+ non-HSCs from the Carnegie Stage 15-16 dorsal aorta (CS15-16) were downloaded from a previous report¹⁴³ (NCBI accession GSE151877). Raw RNA-seq reads from this previous study were aligned and analyzed as described above. Regarding the nomenclature surrounding this dataset: it was previously shown that all HSCs in the human dorsal aorta reside in the CD144+ CD45+ fraction,¹⁵⁶ which we thus define as “HSCs”, in contrast to the CD144+ CD45- fraction, which we refer to as “non-HSCs”.

Computational scripts are available at Github: https://github.com/lohlaboratory/blood-differentiation.

## Single-cell RNA-sequencing of differentiated hPSCs

Using the 10x Genomics Chromium v3 platform,²¹⁰ we performed single-cell RNA-seq on day-0 undifferentiated hPSCs, day-1 posterior primitive streak, day-2 posterior primitive streak, day-3 lateral mesoderm, day-4 artery endothelium, day-7 hemogenic endothelium, and day-10 HLF+ HOXA+ hematopoietic progenitors, all of which were generated from the H7 hPSC line. Libraries were prepared using the Chromium 3' Kit v3 (10x Genomics, 1000075) and Chromium Single Cell Chips (10x Genomics, 1000073) following the manufacturer's guidelines. Libraries were prepared with i7 indices (compatible with Illumina sequencers) and then multiplexed. Multiplexed libraries were sequenced across two lanes of the Illumina NovoSeq 6000 sequencer to obtain 2x150bp paired-end reads (PE150). Unique sequences in each i7 index were used for demultiplexing.

Computational analysis of single-cell RNA-seq data was performed largely as described previously.¹⁰⁴ Cell Ranger (10x Genomics) was used to perform read alignment to the hg38 reference genome, filtering, barcode counting, and unique molecular identifier (UMI) counting. Cell matrix files generated from Cell Ranger were imported into RStudio using the function "Read10x_h5" in Seurat v4¹⁵⁴ to generate a Seurat object. Subsequent processing followed previously described workflows (https://satijalab.org/seurat/articles/pbmc3k_tutorial), which included normalizing data, finding variable features, scaling, and performing linear dimensional reduction to create Uniform Manifold Approximation and Projection (UMAP) graphs.²¹¹ Importantly, all cells within each sample population were shown on the UMAP graph, without any pre-selection of a cell-type of interest.

Computational scripts are available at Github: https://github.com/lohlaboratory/blood-differentiation.

## Comparison of hPSC-derived hematopoietic progenitors generated using various differentiation protocols by single-cell RNA-sequencing

We downloaded published single-cell RNA-sequencing data of hPSC-derived hematopoietic progenitors differentiated using three different protocols that were originally developed by the Keller laboratory⁵⁴ (European Bioinformatics Institute Accession E-MTAB-8205 sample EXP2_CTRL2_4823STD7231845),¹⁴⁴ the Zandstra laboratory (NCBI Accession GSE207157 sample GSM6280645),¹⁴⁵ or the Elefanty, Stanley, and Ng laboratories (Calvanese et al.; NCBI Accession GSE162950 sample GSM6205033).³⁰ Data were processed as described above in the section “Single-cell RNA-sequencing of differentiated hPSCs” using Seurat v4¹⁵⁴ (https://satijalab.org/seurat/articles/pbmc3k_tutorial) to generate UMAP plots.²¹¹ All cells within each of the respective sample populations were shown on the UMAP projection, without any pre-selection of a cell-type of interest.

Computational scripts are available at Github: https://github.com/lohlaboratory/blood-differentiation.

## Comparison of differentiated hPSCs and primary human hematopoietic stem and progenitor cells by single-cell RNA-sequencing

Single-cell RNA-sequencing data of human fetal hematopoietic stem and progenitor cells (HSPCs) were downloaded from a previous report³⁰ (NCBI Accession GSE162950, samples GSM4968832 and GSM4968833). hPSC-derived (in vitro) and human fetal (in vivo)
data were merged and integrated using Seurat v4<sup>154</sup> in accordance with previously described methods (https://satijalab.org/seurat/articles/integration_introduction). In the resultant merged dataset, we annotated different cell-types based on expression of cell-type-specific markers.<sup>30</sup>

Computational scripts are available at Github: https://github.com/lohlaboratory/blood-differentiation.

## Computational analysis of mouse embryo single-cell RNA-sequencing data

Single-cell RNA-sequencing data of E8.5-E11 whole mouse embryos were downloaded from a previous report (Qiu et al., 2023; NCBI Accession GSE186069 and GSE228590).<sup>85</sup> We employed the cell-type annotations defined by the authors of the original study.<sup>85</sup>

Single-cell RNA-sequencing data of E10-E11 mouse hematopoietic progenitor and endothelial cells were downloaded from a previous report (NCBI Accession GSE112642).<sup>28</sup> We employed the cell-type annotations defined by the authors of the original study.<sup>28</sup>

Single-cell RNA-sequencing data of whole E8.5 mouse embryos were plotted using an online browser (https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/).<sup>81</sup> We employed the cell-type annotations defined by the authors of the original study.<sup>81</sup>

Computational scripts are available at Github: https://github.com/lohlaboratory/blood-differentiation.

## Transplantation of human HSPCs or hPSC-derived hematopoietic progenitors into recipient mice

Male and female 8- to 12-week-old NOD-SCID Il2rg-/- (NSG) mice were used as recipient mice. Immediately prior to transplantation, NSG mice received a single dose of 2Gy irradiation. 1-3 million hPSC-derived hematopoietic progenitors, or alternatively, 80,000-100,000 human CD34+ cord blood hematopoietic stem and progenitor cells were transplanted intrafemorally in 20-30 µL IMDM + 20% FBS into irradiated NSG mice. 3-6 months post-transplantation, mice were sacrificed, and bone marrow was analyzed by flow cytometry to detect the presence of human cells. The following antibodies were used to detect human hematopoietic cells in transplanted mice via flow cytometry: BD Horizon V450 anti-human CD45 antibody (BD Biosciences, 560367), APC-Cy7 anti-Human HLA-A/B/C antibody (Biolegend, 311425), PE anti-Human CD33 antibody (Biolegend, 303404), and APC anti-Human CD19 antibody (Biolegend, 302212).

## Intravital bioluminescent imaging

Twenty minutes prior to imaging, mice were injected intraperitoneally with 100 µL of 15 mM AkaLumine HCl (otherwise known as TokeOni [Aobious], which was dissolved in H₂O).<sup>168</sup> Mice were anesthetized using isoflurane and placed in the imaging chamber of a SII Lago-X bioluminescent imaging machine. Imaging parameters were kept constant throughout the duration of each experiment with no images reaching saturation (Binning = 4, FStop = 1.2, exposure time = 10 s). Subsequent image analysis was done in Aura.

## Construction of Efnb2-CreERT2 mice

Pronuclear injection was performed on 1-cell stage mouse zygotes from the C56BL/6 genetic background, to deliver a mixture of sgRNA (5 ng/µL), transgenic donor DNA (5 ng/µL) and Cas9 protein (15 ng/µL). Subsequently, microinjected zygotes were transferred into the oviduct of a pseudopregnant CD1 surrogate mother.

The sgRNA sequence targeting the endogenous mouse *Efnb2* gene was as follows: TCAGACCTTGTAGTAAATGT. The transgenic donor DNA sequence encoding the *GSG-P2A-CreERT2-F5* cassette, including 5' and 3' homology arms targeting the *Efnb2* locus, was as follows:

GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAAC
CCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCTTTTTTGCGGCATT
TTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG
AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT
GTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA
CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG
CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTG
CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCA
GCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC
AGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC
TCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAG
CGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC
CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA
AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTG
TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT
CGGGCTGAACGGGGGGTTCGTGCACACAGCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT
GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG
GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC
GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGAC CGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATT AATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC ACCCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGA CCATATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGATTTAAATTTCCCGGGTCCCATCACTCCT TCCTTTCTGGCGCATCGCTGCTGGTAGGCAGGGCTTCTGTGGAAGGCGGGCGTTTAAAGACGGACATATAACACCCAGTTTCCAGC ATGAGGGAAGAGCAGGCCAGGCCAGGGCTGCACCATGCTCTGGAGTAGCTGCGTGTGCCTTTTTCCAGCCGAGCCTGGTGCAT CACCGGGAAGTCAGAGGCTAGGACGGCCCAAGGGCTTGCTGGCTGTCTCGCCATTCTGCTGCCGGTGACACAGACAAGGAAGC AGTTGGGGATTGTATGAGCATTGGACCTTGAGGCTCCTTTGCCCTGTCTGTCAAGTTCGCTCTGAGGGTTGAGCGTTGGACATGCT TAATTATTACATTTCGGGGTTTCTGCAAGAAACAGGGCACACCCAGGACTCTGGTAACTGGCTCTTCTTGCTCCTGTGCAGGTTCTA GCACCGATGGCAACAGCGCGGGGCATTCGGGAACAATCTCCTGGGTTCCGAAGTGGCCTTATTCGCAGGGATCGCATCAGGAT GCATCATCTTCATCGTCATCATCACACTTTGGTGGTGCTGCTGCTCAAGTACCGCAGGAGACACCGCAAACACTCTCCACAGCAC ACGACCACGCTGTCTCTCAGCACACTGGCCACGCCCAAGCGAGGTGCAACAACAATGGCTCGGAGCCCAGTGACGTTATCATA CCACTAAGGACTGCAGACGCTTCTGCCCGCACTACGAGAAGGTCAGCGGGGACTATGGGCACCCGGTGTACATCGTGCAG GAGATGCCCCCACAGAGTCCTGCAAACATTTACTACAAGGTCGGATCCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAG ACGTGGAGGAGAACCTGGACCTATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGAT GAGGTTCGCAAGAACCTGATGGCATTGTTCAGGATCGCCAGGCGTTTAAATGCTTCTGTCGTTTGCCG GTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTC AGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACC AAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTA GCGTTCGAACGCACTGATTTCGACCAGGTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCT GGGGATTGCTTATAACACCTGTTGATGTAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAA TGTTAACCATATTGGCAGAACGAAAACGCTGTTGATCACCAGCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGT CGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGC CATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGAC TCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATA CCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAAACCTGGATAGTGAAACAGGGGCAAT GGTGCGCCTGCTGGAAGATGGCGATCTCGAGCCATCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGAT CAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCCTTGTGGATGCTGAGCCCCCAT ACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTG GTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTG GCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACA GGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCGCCATGATGAATCT GCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCT GGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTG CAGCAGCAGCACCAGCGGCTGGCCCAAGCTCCTCCTCATCCTCTCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTG TACAGCATGAAGTGCAAGAACGTGGTGCCCTCTATGACCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTACATGCGCCCACT AGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAG TATTACATCACGGGGAGGCAGAGGGTTTCCCTGCCACAGCTTGACGTCGACGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAAA GGTATAGGAACTTCGCTAGGCCGCAGACCTGCGACTCCCAAGGGAACTCGCACCTTGTTCTTGGCACGCAGGGACTGCCTGAGC CTGGCTGTGGGGGCAGGATGCCTCCTGGAAGAGCCTGGATCTGGACAGTTTTGTAGTCTGTAGCTTTTCCGACCCTGGGGACCAC AGACCCTCCCCGGAAGCTGGAAGACTGCTAGGAGATCCCCACTTGGACTGCCGCGGCCCACGCGGACCTCCAAGCCATGCAC CCAGCCACTCAGGCCTCTGCAGAGCCCGGGGAGGACACGGTAGGCTATGGATGGCGCAGCAGCATCTTAGGAGAAGGTTGCGC ACCAGGCCGCCCCTGCCTCCACGTTTCCTGCCGTGCACACTGGACTTATCACTTGGACCTCGGGTTCAGTAAGGTTTTCAAAGA TCTCTAGTGTTTAGTCCTCACTCACTCACTCACTCACTCACTCCTTCTCTTGCCAGGGCTCTGCAGCAAACTCCCTAGACCCCTCAC TCCACGTACTGCATCATTACGGGACACTCACCACAGAGTCCCAAGCTCCACCCTTTACACCAAGATCAAATTAGATGGGTATTAGGT ACAGAAGAACCTGCTTGCCTGGAGGCCGGGTCAGCCGGGAAGCGCAGATGTGTGGAGGAGTGAGGAGGTGCTGGCTGCCACG GCAGGTCAAGGCTGCTTGCCTGCCCTGGAGCATAGTAGGGATGCAGGAAGGAAATAGATAATGCTTTGGTTTTTCTGAGAGGACA GAGACAGGTGGGAGGTGACTGACTGGTGAGTGGTGGGGAGCCTTTCACTACCACACAGCTATGCAGCAGGGAATCAAAAGGCGC GCCTGTACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCG TTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA ACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCG TGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA AACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGC TGATTTAACAAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTG
QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical tests

One factor ANOVA (analysis of variance, alpha = 0.05) was performed to test for statistical significance in Figures 1K-1M. In all other figures, statistical significance was evaluated using a t test (alpha = 0.05). In all figures, the following notation was used to denote statistical significance: \*p < 0.05, \*\*p < 0.01, n.s. = not significant. Histograms and line graphs typically depict the mean, and error bars indicate standard error of the mean (SEM).