GSM6189369: Villous tissue 1, RPL, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
Product of conception (POC) including placental and decidual tissue were collected from 3 patients (Supplementary Table 17) suffering from idiopathic (unexplained) recurrent pregnancy loss defined as 2 or more failed pregnancies where common risk factors were excluded (i.e. obesity, use of alcohol/tobacco/drugs, Mullerian anomaly (septum), balanced translocation, uncontrolled diabetes, uncontrolled hypothyroidism, anti-phospholipid antibody syndrome (or other coagulopathy). All samples had normal karyotype based on clinical cytogenetics testing including chromosomal analysis (GTG banded chromosomes analyzed at the 450-550 band levels) and array comparative genomic hybridization performed in accordance with current International Standing Committee on Human Cytogenetic Nomenclature (ISCN 2009). Prior to single-cell capture, samples were thawed in 10ml of Thawing Medium consisting of DMEM/F-12 (Thermo Fisher Cat No. 11320033) supplemented with 10% FBS and 100 units/mL of penicillin and 100 µg/mL of streptomycin (Thermo Fisher Cat No. 15140122) that was prewarmed in a 37°C bead bath and centrifuged at 300 x g for 8 min. The supernatant was carefully removed without disturbing the cell pellets. The cell pellets were each resuspended in 0.5 mL of Thawing Medium, and the cell suspensions were placed on ice and passed through a prewetted 40-µm nylon mesh cell strainer. Cell suspensions were centrifuged at 300 x g for 8 min at 4°C, and the supernatant was carefully aspirated without disturbing the cell pellets.Each sample was rinsed in PBS before the addition of 5ml of digestion media (MEM/F12 +10% FBS +P/S with 2.0mg/mL collagenase type 1A) and incubated for 50min at 37⁰C. The sample was strained through a pre-wetted 100um filter in a 50ml conical tube and rinsed twice with 5 mL of DMEM/F12 +10% FBS +P/S and centrifuged at 300g for 8 min. The cell pellets were resuspended in 1 mL of cold Recovery Cell Culture Freezing Medium (Thermo Fisher Cat No. 12648010) , and the cell suspension was transferred to a cryogenic storage vial. The cryogenic storage vial was placed in a Corning CoolCell FTS30, which was then placed in a -80°C freezer overnight. Samples were stored at -80°C before being thawed and processed for single-cell RNA-Seq. The cell pellets were resuspended in 100 µL of cold Thawing Medium, and cell count and viability were assessed using 0.4% Trypan Blue and a Countess II automated cell counter. For each sample, Chromium Chip B (10x Genomics Cat No. 1000153) were loaded (Supplementary Table 17). Following cell loading, cDNA and library preparation was performed identically for all samples using the Chromium Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics Cat No. 1000075) according to the manufacturer's protocol. Libraries were sequenced using an Illumina NovaSeq 6000 using 2x94 cycle paired-end.