title: TS21_9_B
organism: Homo sapiens
description: Protocols: Human foetal bone and liver samples were obtained from 15 Ts21 foetuses aged 12-20 pcw and 5 healthy foetuses aged 11-19 pcw, following termination of pregnancy and informed written consent. The human foetal material was provided by the Joint MRC/Wellcome Trust (Grant MR/R006237/1) Human Developmental Biology Resource (​http://www.hdbr.org​), with maternal informed consent, in accordance with ethical approval by the National Health Service (NHS) Research Health Authority, REC Ref: 18/LO/0822. HDBR is regulated by the UK Human Tissue Authoriy (HTA; www.hta.gov.uk) and operates in accordance with the relevant HTA Codes of Practice. Foetal livers and femurs were received in L15 media and processed within 3h from dissection. Livers were cut in smaller pieces with a scalpel and transferred to a tube containing prewarmed digestion media: RPMI (Gibco) supplemented with 10% FBS (Gibco), penicillin/streptomycin (10U/ml penicillin, 100ng/ml streptomycin, Sigma Aldrich), 2mM L-glutamine (Thermo Scientific), 1x MEM NEAA (Gibco), 1mM Sodium pyruvate (Gibco) and 1.6mg/ml Collagenase-IV (Sigma Aldrich). The tube was vortexed for 10”, then incubated at 37ºC for 30 minutes, and vortexed for 10” every 15 minutes. The digested tissue was filtered through a 100μm filter and diluted in cold D-PBS (Gibco). Cells were centrifuged at 300g for 5 minutes, then aliquoted and cryopreserved in KnockOut Serum Replacement (Gibco) + 5% DMSO (Sigma Aldrich). For femurs, adherent material was removed, then the epiphyses were removed with a scalpel and the bone marrow flushed with D-PBS. The remaining bone was cut in small pieces, then ground with a mortar and pestle using digestion media and incubated at 37ºC for 30 minutes, vortexing every 15 minutes. The digested material and the bone marrow flush were mixed and filtered through a 100μm filter. Cells were centrifuged at 300g for 5 minutes, then aliquoted and cryopreserved in KnockOut Serum Replacement (Gibco) + 5% DMSO (Sigma Aldrich). Cells were stored in liquid nitrogen until further analysis.On the day of FACS sorting, cells were rapidly thawed at 37℃ and transferred to complete RPMI media. Live cell enrichment was performed using MACS Dead Cell Removal Kit following the manufacturer's instructions. When depleting for CD235a+ cells, a magnetic negative selection was performed using CD235a Microbeads and MACS LS columns following the manufacturer's instructions.For FACS sorting, ce​lls were stained with Zombie Aqua to exclude dead cells and CD45-FITC (MHCD4501), CD3-AF700 (OKT3), CD19-AF700 (HIB19), CD56-AF700 (BI59), CD8-AF700 (SKI), CD11b-AF700 (CBRM1/5), CD14-AF700 (61D3), CD71-APC (CY1G4), CD235a-APC (GA-RD)for 30 minutes at ​4°C. Cells were centrifuged for 5 minutes at 300 g, 4°C, resuspended in a final volume of 500 μl of 5% FBS in PBS and subsequently filtered into polypropylene FACS tubes. Cells were sorted on a BD FACS Aria Fusion. Immediately after cell enrichment, single cell libraries were prepared on the 10x Chromium with a target of 50,000 cells. Chromium Next GEM Single Cell 3' Kit v3.1, Chromium Next GEM Chip G Single Cell Kit and Dual Index Kit TT Set A were used. Immediately after cell enrichment, single cell libraries were prepared on the 10x Chromium with a target of 50,000 cells. Chromium Next GEM Single Cell 3' Kit v3.1, Chromium Next GEM Chip G Single Cell Kit and Dual Index Kit TT Set A were used.