characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-18T00:22:51Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-18T00:22:51Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112486897
characteristics.characteristic: External Id
characteristics.text: Wellcome Sanger Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-18T00:22:51Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-18T00:22:51Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14604223
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12650:Pla_Camb10123934
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: trophoblast cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: This dataset profiles: A) human Trophoblast stem cell (TSC) lines BTS5 and BTS11 derived by Okae and colleagues(Okae, H. et al. 2018) and B) human primary trophoblast organoids grown and differentiated into EVT as previously described by Turco et al 2018 and Sheridan et al 2020. See details in the \growth protocol\ section below. Both organoids were dissociated for single-cell or single-nuclei RNA-sequencing using the following protocols. - Dissociation of human Trophoblast stem cell: For single-cell transcriptome analysis, TS/EVT cells were PBS washed and dissociated with TrypLE as above. TrypLE was neutralised with PBS supplemented with 10%nFBS instead and filtered through a 40um strainer (pluriSelect pluriStrainer Mini). Cells were pelleted and washed twice with 1mL of PBS supplemented with 0.04% BSA (WAKO) and resuspended in 100µL of the same PBS+BSA before counting with LunaFL. A total of 2,000-5,000 cells were loaded onto the 10x Genomics Chromium inlet. - Dissociation of primary trophoblast organoids (for EVT): Single-cell isolation from EVT organoids was performed as previously described (Sheridan et al 2020). In brief, EVT organoids were released from the ibidi dish with Cell Recovery Solution and washed with ice cold PBS. EVT organoids were dissociated with Stem Pro Accutase at 37C for 5min and 0.5 m/mL Collagenase V solution for 15 min. Single-cell suspension from EVT organoids was resuspended in 0.4%/BSA, filtered through a 40um strainer, stained with trypan blue and counted with a haemocytometer. Single cell suspensions were diluted to targeted final concentration to recover ~5000-10,000 cells per sample prior to loading prior to loading of the 10x Genomics Chromium inlet. - Nuclei of trophoblast organoids (primary and okae): Trophoblast organoids pellets were released from the culture plate as previously described (Sheridan et al 2020). In brief, 1.5ml of Cell Recovery Solution was added into the pellet to dissolve the Matrigel for 45 min on ice. Trophoblast organoids were collected by centrifugation at 600g for 6 min. Supernatant was removed and the pellet was resuspended in ice cold PBS. Trophoblast pellet was snap frozen for single nuclei isolation. Nuclei was released with the dounce homogenizer method as previously described (Krishnaswami SR et al 2016). Nuclei suspension was resuspended in PBS/1% BSA stained with trypan blue, counted with a haemocytometer and diluted to targeted final concentration to recover ~10,000 nuclei per sample prior to loading prior to loading of the 10x Genomics Chromium inlet. Trophoblast stem cell (TSC) lines BTS5 and BTS11 derived by Okae and colleagues were grown as described previously (Okae, H. et al. 2018). In brief, TSC self-renewing medium (TSCM) components were substituted with local suppliers with the exception for 30% w/v BSA from WAKO Japan and CHIR99021 concentration was increased to 6 µM which maintained the undifferentiated morphology as well as preserving its EVT invasive morphology. TSCs were grown on 5 µg/mL Collagen IV (Corning) coated wells and early passaged cells between p24 to 26 were used for differentiation and analysis. For 2D differentiation into EVT identity, cells were seeded at a density of 1.3x105/cm2 (corresponding to 125,000 cells plated on a well of a 6-well plate) in EVT media 1 detailed below supplemented with ice-cold 2% Matrigel GFR (Corning) before seeding on 1 µg/mL Collagen IV (Corning) coated wells (D0). Three days later (D3), media was changed to EVT media 2 supplemented with ice-cold 0.5% Matrigel GFR. Three days later (D6), the media was changed to EVT media 3 supplemented with ice-cold 0.5% Matrigel GFR. Cells were TrypLE-treated for downstream analysis 48h later (D8). For CXCL16 induction experiments, a final concentration of 100ng/mL CXCL16 (RnD 976-CX-025 with carrier, dissolved in 0.1%BSA(WAKO)/PBS) were supplemented to EVT media 2 or 3 and analysed 48h later. The induction was controlled by supplementing an equal volume of 0.1%BSA/PBS. In total, six trophoblast organoids were grown and differentiated into EVT as previously described (Turco et al 2018; Sheridan et al 2020). To differentiate trophoblast organoids into EVT, organoids were cultured with trophoblast organoid media (TOM) for ~3-4 days and transferred into EVT media 1 (+NRG1) for ~4-7 days. Once trophoblasts initiate their commitment into EVT (spike emergence), EVT media 2 (-NRG1) is added for 4 days. Donors were differentiated and collected in batches of three that were multiplexed on the same 10x Genomics reaction. Samples for donors 1, 2 and 3 were collected at 3 hours (h), 24h and 48h after the addition of EVTM media 2, while samples for donors 4, 5 and 6 were collected at 48h before, and then 0h, 48h and 96h after, addition of EVTM media 2. Organoids grown in trophoblast organoid media (TOM) media were also collected as a control at 96h. Media compositions were described previously: TSCM = DMEM/F12 with Glutamax (Gibco) supplemented with 0.2% v/v FBS (Gibco), 0.3% w/v BSA (WAKO), 1% ITS-X (Gibco), 2.5 µg/mL L-ascorbic acid-2-phosphate (Sigma), 50 ng/mL EGF (Peprotech AF-100-15), 6 µM CHIR99021 (Tocris 4423), 0.5 µM A83-01 (Tocris 2939), 1 µM SB43154 (Tocris 1614), 0.8 mM VPA (Sigma, dissolved in H2O) and 5 µM Y27632 (Millipore 688000). TOM = Advanced DMEM/F12, N2 supplement (at manufacturer’s recommended concentration), B27 supplement minus vitamin A (at manufacturer’s recommended concentration), Primocin 100 μg/mL, N-Acetyl-L-cysteine 1.25 mM, L-glutamine 2 mM, recombinant human EGF 50 ng/mL, CHIR99021 1.5 µM, recombinant human R-spondin-1 80 ng/mL, recombinant human FGF-2 100 ng/mL, recombinant human HGF 50 ng/mL, A83-01 500 nM, prostaglandin E2 2.5 µM, Y-27632 5 µM. EVT media 1 = Advanced DMEM/F12 (or DMEM/F12 for TSC-EVTM 2D), L-glutamine 2 mM, 2-mercaptoethanol 0.1 mM, penicillin/streptomycin solution 0.5% (vol/vol), BSA 0.3% (vol/vol, WAKO), ITS-X supplement 1% (vol/vol), NRG1 (Cell Signaling 5218SC) 100 ng/mL, A83-01 7.5 µM, Knockout serum replacement 4% (vol/vol) EVT media 2 = Advanced DMEM/F12 (or DMEM/F12 for TSC-EVTM 2D), L-glutamine 2 mM, 2-mercaptoethanol 0.1 mM, penicillin/streptomycin solution 0.5% (vol/vol), BSA 0.3% (vol/vol, WAKO), ITS-X supplement 1% (vol/vol), A83-01 7.5 µM, Knockout serum replacement 4% (vol/vol) ie: EVT medium cat. no. 1 without NRG1. Store the medium at 4°C for up to 1 week. EVT media 3 = DMEM/F12 (for TSC-EVTM 2D), L-glutamine 2 mM, 2-mercaptoethanol 0.1 mM, penicillin/streptomycin solution 0.5% (vol/vol), BSA 0.3% (vol/vol, WAKO), ITS-X supplement 1% (vol/vol), A83-01 7.5 µM, ie: EVT medium cat. no. 1 without NRG1 nor knockout serum replacement. Store the medium at 4°C for up to 1 week. Organoids were enzymatically digested in 70 ml 0.2% trypsin 250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma E9884) in PBS with stirring at 37 °C for 9 min. The disaggregated cell suspension was diluted with medium and passed through a 100-μm cell sieve (Corning, 431752). The disaggregated cell suspension was diluted with medium and passed through a 100-μm cell sieve (Corning, 431752). Cells obtained from both enzyme digests were pooled together and passed through a 100-μm cell sieve (Corning, 431752) and washed in Ham’s F12. The flow-through was centrifuged and resuspended in 5 ml of red blood cell lysis buffer (Invitrogen, 00-4300) for 10 min. Cells were loaded according to the manufacturer’s protocol for the Chromium Single Cell 3′ Kit v3.1 (10X Genomics). Library preparation was carried out according to the manufacturer’s protocol to attain between 2,000 and 10,000 cells per reaction.
characteristics.characteristic: description
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: trophoblast organoid medium
characteristics.characteristic: growth condition
characteristics.text: Primary Trophoblast Organoids
characteristics.characteristic: in vitro system
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: embryo
characteristics.characteristic: organism part
characteristics.text: scRNA-seq
characteristics.characteristic: protocol
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Pla_Camb10123934
characteristics.characteristic: title