title: GSM6027425: scRNAseq 10X, Myometrium, Control, Rep 3, index2C; Mus musculus; RNA-Seq
library_construction_protocol: Dams were euthanized on 17.5 dpc and the reproductive tissues (uterus, decidua, and cervix) were collected. Tissues collected for the preparation of single-cell suspensions were placed in sterile 1X PBS. Immediately following the collection of the cervix, the tissue was mechanically dissociated and enzymatically digested using Collagenase A (160 mg/mL) (Sigma Aldrich, St. Louis, MO, USA) and incubated at 37ºC. Then, the dissociated cells were pelleted by centrifugation at 16,000 x g for 10 min at 20ºC and resuspended with 0.05% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA) prior to a second round of mincing and incubation in 0.05% trypsin-EDTA at 37ºC. The enzymatic reaction was stopped by the addition of FBS (Fetal Bovine Serum, Thermo Fisher). Cells were then filtered using a 70µm cell strainer (Miltenyi Biotec, San Diego, CA, USA) and pelleted by centrifugation at 300 x g for 10 min. Erythrocytes were removed using ACK Lysing Buffer (Life Technologies, Grand Island, NY, USA). Finally, the cells were resuspended in 0.04% Bovine Serum Albumin (BSA) (Sigma Aldrich) diluted in 1X PBS and filtered through a 30µm cell strainer (Miltenyi Biotec). The cell concentration and viability were determined using an automatic cell counter (Cellometer Auto 2000, Nexcelom Bioscience, Lawrence, MA, USA).  and the Dead Cell Removal Kit (Miltenyi Biotec) was used to remove dead cells to obtain a cell viability of ≥80%. Immediately following tissue collection, the uterus and decidua were dissociated to prepare single-cell suspensions. The tissues were mechanically dissociated and enzymatically digested by incubating at 37ºC using enzymes from the Umbilical Cord Dissociation Kit (Miltenyi Biotec). A second round of mechanical dissociation was performed using the gentleMACS Dissociator (Miltenyi Biotec), and dissociated cells were rinsed with 1X PBS (Thermo Fisher Scientific), NY, USA) prior to filtration using a 100µm cell strainer (Miltenyi Biotec). Filtered cells were pelleted by centrifugation at 300 x g for 5 min, erythrocytes were eliminated using ACK Lysing Buffer (Life Technologies), and the cells were rinsed in 0.04% BSA (Sigma Aldrich) and 0.5 mM EDTA (Sigma Aldrich) diluted in 1X PBS. Finally, the cells were filtered using a 30µm cell strainer (Miltenyi Biotec), and the Dead Cell Removal Kit was used to remove dead cells to obtain a cell viability of ≥80%. Generation of gel beads-in-emulsion (GEMs) and preparation of library constructs was performed on viable single-cell suspensions using the 10x Genomics Chromium Single Cell 3' Gene Expression Version 3.1 Kit (10x Genomics, Pleasanton, CA, USA), according to the manufacturer's instructions. Briefly, viable single cells were encapsulated in partitioning oil together with a single Gel Bead with barcoded oligonucleotides within the Chromium Controller. Reverse transcription of mRNA into complementary (c)DNA was performed using the Veriti 96-well Thermal Cycler (Thermo Fisher Scientific, Wilmington, DE, USA). Dynabeads MyOne SILANE (10x Genomics) and the SPRIselect Reagent (Beckman Coulter, Indianapolis, IN, USA) were used to purify resulting cDNA, which was optimized by enzymatic fragmentation, end-repair, and A-tailing. Next, adaptors and sample index were incorporated by ligation. The sample index PCR product was then amplified using the Veriti 96-well Thermal Cycler and double-sided size selection was performed using the SPRIselect Reagent kit. Following the formation of cDNA and final library construct, the Agilent Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, Wilmington, DE, USA) was used determine sample integrity and concentration.