title: scRNA-seq of first and second trimester human reproductive tract
design: scRNA-seq of first and second trimester human reproductive tract
library_construction_protocol: We used the previous protocol optimised for gonadal dissociation and available at protocols.io136. In short, tissues were cut into <1 mm3 segments before being digested with a mix of Trypsin-EDTA 0.25% and DNaseI (0.1mg/ml) for 5-15 minutes at 37°C with intermittent shaking. Samples > 17 PCW were digested using a combination of collagenase and Trypsin/EDTA, a protocol adapted from Wagner et al.,136,137. In short, samples were first digested with a mix of Collagenase 1A (1 mg/ml), DNaseI (0.1 mg/ml) and Liberase TM (50 µg/ml) for 45 minutes at 37°C with rotation. Cell solution was further digested with Trypsin/EDTA 0.25% for 10 minutes at 37°C with rotation. In both protocols, 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 and resuspended in PBS-BSA 0.04% prior to cell counting.  For the scRNA-seq experiments, cells were loaded according to the manufacturer's protocol for the Chromium Next GEM Single Cell 5′ v2 (DUAL) Kit  (10X Genomics) to attain between 2,000 and 10,000 cells per reaction. Library preparation was carried out according to the manufacturer's protocol. 