GSE175498
Single Cell RNA-Seq of the embryonic day 14.5 genital tubercle
Characterize the cellular diversity of the embryonic genital tubercle before sexual dimorphic morphogenesis in male and female mice. Overall design: Genital tubercles were harvested from pregnant female mice on embryonic day 14.5.  Embryos were dissected out in ice-cold Dulbecco's phosphate-buffered saline and a small amount of tissue was taken for genotyping. The embryo was placed into RPMI 1640 medium (Thermo Fisher Scientific, 11875093) on ice for 2 hours while the genotyping for sex took place. After genotyping results were finished three male and three female embryos were chosen from 2 litters and pooled by sex. The genital tubercles were then dissected from the body and placed into 1.3mg/ml collagenase XI (Sigma-Aldrich, C7657-25MG) on ice. Collagenase XI was made by first dissolving 13mg into 1ml TESCA buffer at 37°C for 5 minutes and then further diluting to 1.3mg/ml in 10% inactivated fetal bovine serum/ Dulbecco's phosphate-buffered saline. The genital tubercles were chopped into small pieces using forceps while in 500µl collagenase XI on ice. The entire 500µl of collagenase XI and tissue was collected and placed into a 1.5ml microcentrifuge tube for remainder of digestion.  Cells were agitated at 37°C for 30 mins and tritrated with a glass pipettes every 5 mins. After digestion, cells were centrifuged at 500rpm for 5 minutes at 4°C, then were was resuspended in 100ul of magnesium and calcium free 1xPBS with 0.04% bovine serum albumin, filtered through a 40µm strainer (Flowmi Bel-Art H13680-0040), and placed on ice.  Male and female samples were loaded separately for encapsulation, synthesis and amplification of barcoded cDNAs (10X Next GEM Single Cell 3' Gel Bead Kit v3.1). After QC, Illumina libraries (Next GEM SC 3' Library Kit v3.1) were sequenced on an Illumina NexSeq 500/550 using 2x150bp paired-end reads at a targeted depth of 25K reads per cell.    Libraries were prepared using Chromium Single Cell Library kit V3 (10x Genomics) following standard protocol and sequenced on an Illumina NextSeq using 150 bp paired-end sequencing.