Illumina NovaSeq 6000 sequencing; scRNA-seq of first and second trimester human gonads - 10x dataset
scRNA-seq of first and second trimester human gonads - 10x dataset
FCA_GND8715408_p
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.  We used the previous protocol optimised for gonadal dissociation and available at protocols.io.  https://www.protocols.io/view/human-embryonic-gonad-dissociation-with-trypsin-ed-66fhhbn 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.      library For the scRNA-seq experiments, cells were loaded according to the manufacturer's protocol for the Chromium Single Cell 5′ Kit v.1.0, v1.1 and v.2 (10X Genomics) to attain between 2,000 and 10,000 cells per reaction. Library preparation was carried out according to the manufacturer's protocol. Library preparation was carried out according to the manufacturer's protocol for the Chromium Single Cell 5′ Kit v.1.0, v1.1 and v.2 (10X Genomics).