Multiome scRNA- and scATAC-seq of healthy and trisomy 21 (Down's syndrome) foetal liver
Multiome scRNA- and scATAC-seq of healthy and trisomy 21 (Down's syndrome) foetal liver
Human foetal liver samples were obtained from 3 Ts21 foetuses and 3 healthy foetuses, 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 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). 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. For FACS sorting, ce​lls were stained with Zombie Aqua to exclude dead cells and CD45-FITC (MHCD4501) 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.Nuclei preparation was performed following 10x genomics recommendations. Sorted cells were centrifuged at 300g for 10' at 4ºC. Pellets were resuspended in 45ul chilled lysis buffer (), and incubated on ice for 5'. 50ul of chilled wash buffer () was added and nuclei were centrifuged at 500g for 7' at 4ºC. After removing the supernatant, the nuclear pellet was washed with 45ul chilled diluted nuclei buffer (1x) without pipetting. Nuclei were centrifuged at 500g for 10' at 4ºC. Nuclei were resuspended in 7ul of chilled diluted nuclei buffer and counted. 15,000 nuclei were targeted for library preparation. Immediately after cell enrichment, single nuclei libraries were prepared on the 10x Chromium with a target of 15,000 nuclei. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle and Chromium Next GEM Chip J Single Cell Kit were used. Immediately after cell enrichment, single nuclei libraries were prepared on the 10x Chromium with a target of 15,000 nuclei. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle and Chromium Next GEM Chip J Single Cell Kit were used.