title: Illumina NovaSeq 6000 sequencing; Mapping the developing human immune system across organs - scRNA-seq
design: Mapping the developing human immune system across organs - scRNA-seq
library_construction_protocol: MS5 line transduced with human DLL4 was obtained from G. Crooks (UCLA) as a gift. The MS5-hDLL4 cells were cultured in DMEM (Gibco) with 10% FBS. Two iPSC lines were used in this study. Cell lines HPSI0114i-kolf_2 (Kolf) and HPSI0514i-fiaj_1 (Fiaj) were obtained from the Human Induced Pluripotent Stem Cell initiative (HipSci: www.hipsci.org) collection. All iPSC lines were cultured on vitronectin (diluted 1:25 in PBS; Gibco) coated plates, in TeSR-E8 media (Stemcell Technologies). 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, 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 and checking of ATO, a cell scraper was used to detach ATOs from cell culture insert membranes and detached ATOs were then submerged in cold flow buffer. 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 week 3 ATO cells, and live (DAPI–) cells were sorted for week 5 and week 7 ATO cells before loading onto each channel of the Chromium chip from Chromium single cell V(D)J kit (10X Genomics). For scRNA-seq experiments, either Chromium single cell 3' reagent kit or Chromium single cell V(D)J kits from 10x Genomics were used. Unsorted, or DAPI-CD45+ or DAPI-CD45- FACS-isolated cells were loaded onto each channel of the Chromium chip following the manufacturer's instructions before droplet encapsulation on the Chromium controller. Single-cell cDNA synthesis, amplification, gene expression (GEX) and targeted B cell receptor (BCR) and T cell receptor (TCR) libraries were generated. Targeted enrichment for γδTCR was done following the TCR enrichment protocol from 10x with customised primers binding to the constant region of the TRD and TRG genes as described in Mimitou et al. 2019.
attributes.tag: Experimental Factor: time
attributes.value: 7
attributes.tag: Experimental Factor: immunophenotype
attributes.value: live cell