Gel bead kits v3

Cells were thawed, stained with FACS antibodies and sorted on a Becton Dickinson Aria III or Fusion 2 as described above and as per recommendations in the 10x Genomics single cell protocols. 15,000-24,000 Lin-CD34+ cells were FACS sorted from each sample, and processed as described in (Psaila et al., 2020 (link)). Samples were processed according to the 10x Genomics protocol using the Chromium Single Cell 3′ library and Gel Bead Kits v3 (10x Genomics). Cells and reagents were prepared and loaded onto the chip and into the Chromium Controller for droplet generation. RT was conducted in the droplets and cDNA recovered through demulsification and bead purification. Pre-amplified cDNA was used for library preparation, multiplexed and sequenced on a HiSeq 2500.

RNA from sorted GFP+ thrombocytes and RFP+ thrombocytes were prepared according to 10x Genomics protocol. Sequencing was performed after successful library preparation and quality control. Briefly, the sorted cells were loaded immediately onto 10x Genomics Chromium Chip B. The barcoded cDNA libraries were generated using 10x Genomics Chromium Single Cell 3’ GEM library and Gel Bead Kits V3 according to the manufacturer’s instructions. The library sequencing on Illumina Hiseq 2500 V3 was performed on a depth of a minimum of 20,000 read pairs/cell for each library, and Cell Ranger software (3.0.0) version was used for barcode recovery [14 (link)–16 ].

Cryopreserved cells pooled from 15 organoids from 3 differentiations from both VEGFA and VEGFA + C protocols were processed for single-cell sequencing as described in Supplementary Methods, and processed using the Chromium Single-Cell 3′ library and Gel Bead Kits v3.1 (10X Genomics) as per kit instructions. Demultiplexed FASTQ files were aligned to the human reference genome (GRCh38/hg38) using standard CellRanger (version 6.0.1) “cellranger count” pipeline (10X Genomics). SingCellaR (ref. 31 (link); https://supatt-lab.github.io/SingCellaR.Doc/) was used for the downstream analysis.