Illumina NovaSeq 6000 paired end sequencing; scATAC-seq of first and second-trimester human gonads
scATAC-seq of first and second-trimester human gonads
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. Human embryo and fetal samples were obtained from the MRC and Wellcome-funded Human Developmental Biology Resource (HDBR43, http:// www.hdbr.org), with appropriate maternal written consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee (08/H0906/21+5). 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.   All tissues for sequencing and spatial work were collected in saline (HypoThermosol biopreservation media) and stored 4°C until processing. Tissue dissociation was conducted within 24 hours of tissue retrieval with the exception of tissues that were cryopreserved and stored in -80°C.  We used the previous protocol optimised for gonadal dissociation 5 and available at protocols.io    In short, tissues were cut into <1 mm segments before being transferred to a 15ml tube. Tissues were digested with Trypsin/EDTA 0.25% between 5-15 minutes at 37°C with intermittent shaking. Digested tissue was passed through a 100 µm filter, and cells collected by centrifugation (500 x g for 5 minutes at 4°C). Cells were washed with flow buffer (PBS containing 5 % (v/v) FBS and 2 mM EDTA) prior to cell counting. Single-nuclei suspension was produced from dissociated cells for doing scATAC-seq, following the manufacturers' instructions, and from frozen tissue sections for doing the multiomic snRNA-seq/scATAC-seq. For the scATAC-seq experiments, cells were loaded according to the manufacturer's protocol for the Chromium Single Cell ATAC v1.0 and v to attain between 2,000 and 10,000 cells per well. Library preparation was carried out according to the manufacturer's protocol.