title: scRNA-seq of T cell differentiation in Artificial Thymic Organoids
design: scRNA-seq of T cell differentiation in Artificial Thymic Organoids
library_construction_protocol: We followed the PSC-ATO protocol as previously described30. iPSC cells were harvested as a single-cell suspension and seeded (3×106 cells per well) in GFR reduced Matrigel (Corning) - coated 6-well plates in X-VIVO 15 media (Lonza), supplemented with rhActivin A, rhBMP4, rhVEGF, rhFGF (all from R&D Systems), and ROCK inhibitor (Y27632; LKT Labs) on day -17, and only rhBMP4, rhVEGF and rhFGF on days -16 and -15. Cells were harvested 3.5 days later (day-14) , and isolated by FACS for CD326-CD56+ (PE anti-human CD326 antibody, Biolegend, 324205; APC anti-human CD56 antibody, Biolegend, 318309) human embryonic mesodermal progenitors (hEMPs).  Isolated hEMPs were combined with MS5-hDLL4 at a ratio of 1:50. Two or three cell-dense droplets (5×105 cells in 6 μl hematopoietic induction medium) were deposited on top of an insert in each well of a six-well plate. Hematopoietic induction medium composed of EGM2 (Lonza) supplemented with ROCK inhibitor and SB blocker (TGF-β receptor kinase inhibitor SB- 431542; Abcam) was added into the wells outside the inserts so that the cells sat at the air-liquid interface. The organoids were then cultured in EGM2 with SB blocker for 7 days (days -14 to -7), before the addition of cytokines rhSCF, rhFLT3L, rhTPO (all from Peprotech) between days -6 to 0. These 2 weeks formed the hematopoietic induction phase. On day 1, media was changed again to RB27 (RPMI supplemented with B27 (Gibco), ascorbic acid (Sigma-Aldrich), penicillin/streptomycin (Sigma-Aldrich) and glutamax (Thermo Fisher Scientific)) with rhSCF, rhFLT3L and rhIL7. The organoids can be maintained in culture for 7 more weeks in this medium. For dissociation of organoids on day -7, they were removed from culture insert and incubated in digestion buffer, which consisted of collagenase type IV solution (StemCell Technologies) supplemented with 0.88mg/ml collagenase/dispase (Roche) and 50U DNase I (Sigma), for 20 minutes at 37°C. Vigorous pipetting was performed in the middle of the incubation and at the end. After complete disaggregation, single cell suspension was prepared by passing through a 50-μm strainer.  For dissociation of organoids from day 0 onwards, a cell scraper was used to detach ATOs from cell culture insert membranes and detached ATOs were then submerged in cold flow buffer (PBS (Gibco) containing 2% (v/v) fetal bovine serum (FBS; Gibco) and 2 mM EDTA (Invitrogen)). Culture inserts were washed and detached ATOs were pipetted up and down to form single-cell suspension before passing through a 50-μm strainer.   Cells were then stained with designed panels of antibodies and analyzed by flow cytometry. FACS was performed at the same time and live human DAPI-anti-mouse CD29- (APC/Cy7 anti-mouse CD29 antibody, Biolegend, 102225) cells were sorted for day -7, day 0 and week 3 samples, and live (DAPI-) cells were sorted for week 5 and week 7 samples before loading onto each channel of the Chromium chip from Chromium single cell V(D)J kit (10X Genomics). The metadata for all the ATO samples can be found in ATO_manifest.csv. For the day -14 sample, some sorted (both hEMP and the rest of DAPI- fraction) and unsorted cells were stained with hashtag antibodies (TotoalSeq-C antibodies from Biolegend, see ATO_hashtagging.csv, following 10X cell surface protein labeling protocol) before being mixed together with some mouse stromal cells (MS5-hDLL4) for 10X loading. For week 1 sample, hashtag antibodies were added in at the same time as the FACS antibodies i.e. before sorting. Single-cell cDNA synthesis, amplification and gene expression (GEX) and cell surface protein (CITE-seq) libraries were generated following the manufacturer's instructions. Single-cell cDNA synthesis, amplification and gene expression (GEX) and cell surface protein (CITE-seq) libraries were generated following the manufacturer's instructions.