Samples
Fetuses were obtained after voluntary terminations of pregnancy, which were performed either via medical or surgical procedures. The termination methods used did not compromise the integrity or morphology of the fetuses analysed in this study. Only well-preserved fetuses, without evidence of structural damage, were included. All tissue samples used for this study were obtained with written informed consent from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. The human embryonic and fetal material was provided by the Joint MRC–Wellcome Trust (grant number MR/R006237/1 and MR/X008304/1) Human Developmental Biology Resource (HDBR, http://www.hdbr.org), with appropriate maternal written consent and approval from the Fulham Research Ethics Committee (REC reference 18/LO/0822 and 23-LO/0312) and Newcastle & North Tyneside 1 Research Ethics Committee (REC reference 18/NE/0290). The HDBR is regulated by the UK Human Tissue Authority (HTA; www.hta.gov.uk) and operates in accordance with the relevant HTA Codes of Practice. This research was also supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.
Assignment of developmental stage
Embryos up to 8 PCW were staged using the Carnegie staging method62. At stages beyond 8 PCW, age was estimated from measurements of foot length and heel-to-knee length and compared with the standard growth chart63. A piece of skin or, where this was not possible, chorionic villi tissue was collected from every sample for quantitative PCR analyses using markers for the sex chromosomes and the autosomes 13, 15, 16, 18, 21 and 22, which are the most commonly seen chromosomal abnormalities. All samples were karyotypically normal.
Tissue processing
All tissues for sequencing and spatial work were collected in HypoThermosol FRS Preservation solution (Sigma-Aldrich) and stored at 4 °C until processing. Tissue dissociation was conducted within 24 h of tissue retrieval with the exception of tissues that were cryopreserved and stored at −80 °C (Supplementary Table 1).
We used a previously described protocol optimized for gonadal dissociation and available at protocols.io64. In brief, tissues were cut into <1 mm3 segments before digestion with a mix of trypsin–EDTA 0.25% and DNase I (0.1 mg ml–1) for 5–15 min at 37 °C with intermittent shaking. Samples >17 PCW were digested using a combination of collagenase and trypsin–EDTA using a previously described protocol64,65, but with modifications. In brief, samples were first digested with a mix of collagenase 1A (1 mg ml–1), DNase I (0.1 mg ml–1) and Liberase TM (50 µg ml–1) for 45 min at 37 °C with rotation. The cell solution was further digested with trypsin–EDTA 0.25% for 10 min at 37 °C with rotation. In both protocols, digested tissue was passed through a 100 µm filter and cells were collected by centrifugation (500g for 5 min at 4 °C). Cells were washed and resuspended in PBS–BSA 0.04% before cell counting.
Single-nucleus suspension
Single-nucleus suspensions were isolated from dissociated cells when performing scATAC–seq, following the manufacturers’ instructions, and from frozen tissue sections when performing multi-omic snRNA-seq and scATAC–seq. For the latter, thick (300 µm) sections were cryosectioned and kept in a tube on dry ice until subsequent processing. Nuclei were released by Dounce homogenization as described in detail in the methods at protocols.io (https://doi.org/10.17504/protocols.io.bp2l6n1xkgqe/v1).
Tissue cryopreservation
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