title: Illumina NovaSeq 6000 sequencing; Spatial transcriptomics (Visium) of first and second-trimester human gonads
design: Spatial transcriptomics (Visium) of first and second-trimester human gonads
library_construction_protocol: 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  Ten micron cryosections were cut and placed in duplicate on Visium slides (beta product version). These were processed according to the manufacturer's instructions. Briefly, sections were fixed with cold methanol, stained with haematoxylin and eosin and imaged on a Hamamatsu NanoZoomer S60 before permeabilisation, reverse transcription and cDNA synthesis using a template-switching protocol.   Second-strand cDNA was liberated from the slide and single-indexed libraries prepared using a 10x Genomics PCR-based protocol.