Cryostor cs10

After aspirating media from feeder cells, 1 mL of Epstein–Barr virus (EBV, ATCC, #VR-1492) was added per flask with 1–5 × 10⁶FANCA c.3934 + 2T > C (rs771775516) PBMCs and 3 mL of LCL media (below) under normoxic conditions (21% O₂, 5% CO₂, 37 °C). At 10–14 days post transformation, cells were removed from feeder cells by transferring to a new T-25 flask. Once transformation reached later stages (increased turbidity or yellowing of culture media) cells were transferred from a T-25 to T-75 flasks. Cells were kept at a density of 1–3 × 10⁵ cells/mL and split to additional T-75 flasks as needed. Once 2e7 cells were reached, aliquots were frozen at a density of 5e5 cells/mL in CryoStor CS10 (StemCell Technologies, Vancouver, CA, USA, #07930). An aliquot of 1 × 10⁵FANCA c.3934 + 2T > C (rs771775516) LCLs were then stained with PE anti-human CD19 antibody clone HIB19 (BioLegend, San Diego, CA, USA, 302254) for flow cytometry.

Hepatic fibroblast derived iPS line ATCC‐HYS0103 Human Induced Pluripotent Stem (IPS) Cells were purchased directly from the vendor. hESC‐Qualified Matrigel (Corning 354277) was used coat culture dishes for feeder‐free expansion of induced pluripotent stem cells and were routinely cultured and maintained in mTeSR1 (STEMCELL Technologies, 85850) in a humidified incubator at 37 °C with 5% CO₂. Cells were passaged once a week using selective dissociation reagent ReLeSR (STEMCELL Technologies 05872) and seeded using the cell aggregate counting method described in “Plating Human ES and iPS Cells Using the Cell Aggregate Count Method” (Appendix 1) from the STEMCELL Technologies—“Maintenance of Human Pluripotent Stem Cells in mTeSR1” technical handbook to assess the size of aggregates and seed them in low, medium or high densities, as described. All cryopreservation of cells was performed in CryoStor CS10 (STEMCELL Technologies 07930) freezing media.
For the glass controls in the experiments—sterile rh‐Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro Accutase (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10 µm Rock inhibitor Y27632 (ATCC ACS‐3030).

For scATAC-seq library construction, the samples were cryo-preserved with CryostorCS10 (STEMCELL). The nuclei were extracted according to the 10x Genomics user guide. Cryopreserved samples were recovered in prewarmed media (RPMI 1640 + 10% FBS) and then subjected to centrifugation at 300×g for 5 min at 4 °C. After DPBS washing, the cells were lysed in chilled lysis buffer (10 mM pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% digitonin, 1% BSA) with gentle pipette mixing and incubation on ice for 3 min. After washing with chilled wash buffer (10 mM pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, and 1% BSA), the nuclei were resuspended in chilled diluted nuclei buffer (10x Genomics) and filtered through 40-μm strainers. The quality and quantity of nuclei were manually examined by trypan blue staining. Nuclei suspensions with a target recovery of 3000-6000 nuclei were subjected to Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 (10x Genomics) according to the manufacturer’s instructions. The library was processed on the Illumina NovaSeq6000 platform for sequencing with 50 bp paired-end reads.

STEMspan-ACF and STEMdiff-APEL Media, STEMspan Megakaryocyte Expansion Supplement, mTeSR1, Dispase, and CryoStor CS10 were purchased from STEMCELL Technologies. BMP4 was from HumanZyme. All other cytokines were obtained from PeproTech. Y-27632 was purchased from Stemgent. Human Collagen IV was from Advanced BioMatrix. Matrigel and antibodies for flow cytometry were obtained from BD Biosciences. I-BET 151(GSK1210151A) was purchased from ChemieTek. MMP-8 Inhibitor I (CAS 236403-25-1) was purchased from Millipore. StemPro Accutase was from Life Technologies. Heparin was purchased from Sigma.