GSM7773150: SCG10; Mus musculus; RNA-Seq
Dissected uterine tissues were thoroughly minced and stored in 90% fetal bovine serum (FBS) (R&D Systems, cat. #S11150) and 10% DMSO (Sigma-Aldrich, cat. #D8418) in liquid nitrogen until the day of processing. scRNA-Seq data was collected over 3 separate experimental batches and 10 pools. For non-pregnant and GD5.5 IIS samples, each replicate consisted of uteri from 2 mice pooled together. At the time of processing, tissues were thawed, washed twice in PBS, and enzymatically disaggregated with Liberase TM (Roche, cat. #5401127001; 0.28 WU/ml), DNase I (Roche, cat. #1010415901; 30 μg/ml) and trypsin (ThermoFisher, cat. #15090046; 0.05%) in Ca++/Mg++-containing HBSS for 60 minutes at 37°C with trituration every 10 minutes. 5 mM EDTA (Life Technologies, cat. #15575020) was added for the final 15 minutes of the incubation. The cell suspensions were filtered through 100 μm filters and checked for viability. All sequencing experiments were performed on samples with a minimum of 70% viable cells, as determined by a Nexcelom Cellaca MX cell counter. Cells were washed in PBS with 0.04% BSA and then incubated with lipid-conjugated oligonucleotides for cell hashing (10X Genomics) for 5 minutes at room temperature. Cells were then washed twice with PBS with 1% BSA. 125,000 cells per sample were multiplexed and, from these pools, 340,000 cells were loaded onto the Chromium Controller (10X Genomics). Samples were processed for single-cell encapsulation and cDNA library generation using the Chromium Single Cell 3' v3.1 Reagent Kits (10X Genomics). Libraries were subsequently sequenced on an Illumina NovaSeq 6000 (Illumina). All samples were sequenced at a sequencing depth of 30,000 reads per cell. Libraries were generated using the Chromium Single Cell 3' v3.1 Reagent Kits (10X Genomics)