title: GSM8823453: endometrium, first trimester decidua, rep2-1; Homo sapiens; RNA-Seq
library_construction_protocol: Endometrial biopsies were performed with the Endocell®, a disposable endometrial cell sampler (Wallach Surgical Devices, Trumbull, CT, USA) in the secretory phase. For collection of endometrial deciduae, women scheduled for an elective termination of an uncomplicated pregnancy in the first trimester at 6-8 weeks estimated gestational age were eligible for inclusion. After dilation and curettage, tissue samples were floated in phosphate buffered saline (PBS, pH 7.2) and the endometrial decidua was isolated from the gestational sac and chorionic villi by mechanical separation using forceps. Endometrial tissue was placed in a tissue culture dish and rinsed with ice-cold PBS to remove blood and mucus. Using fine forceps, each sample was separated such that approximately 100 mg endometrial tissue was fixed in formalin and stored at room temperature for immunohistology; approximately 100 mg was preserved in RNAprotect (Qiagen, 76160) and was placed on ice, and the remainder minced and processed within one hour of collection for scRNA-seq. Cells were isolated from minced endometrial tissues for scRNA-seq by incubation at 37°C in digestion media (DMEM/F12, 3% charcoal-stripped fetal bovine serum (FBS), 1 mg/mL collagenase A (Roche, #11088793001), 0.1 mg/mL DNAse type I (Roche, #10104159001) with agitation on a benchtop shaker at 250 rpm for 15 minutes. The tissue was then further dissociated by repeatedly passing it through a 16G needle attached to a 10 mL syringe. The shaking and needle passage steps were repeated once more to ensure thorough dissociation. The cell suspension was centrifuged at 300 ×g for 5 minutes. The resulting pellet was resuspended in 1 mL of TrypLE Select Enzyme (Thermo Fisher #12563011) containing 0.1 mg/mL DNase I and incubated at 37°C on a shaker at 250 rpm for 15 minutes. 10 mL of ice-cold digestion media was added, and the cell suspension was filtered through a 40 µm cell strainer. Red blood cells (RBC) were lysed using RBC lysis buffer (ThermoFisher, #00-4333-57) and dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec, #130-090-101). The resulting single-cell suspension was used for Chromium (10X Genomics) sequencing. For scRNA-seq, libraries were generated and sequenced using the 10X Chromium Single Cell 3ʹ GEM kit (10X Genomics, v2). Paired end, 2 x 75 bp, sequencing was performed on an Illumina NextSeq 500 instrument (RRID:SCR_014983). Output was demultiplexed and converted to FASTQ format with Cell Ranger (10X Genomics, v3.1.0).