scRNAseq of chorioamniotic membranes
Immediately after delivery, a placental sample was collected at the Detroit Medical Center, Wayne State University School of Medicine (Detroit, MI). Cells from chorioamniotic membranes were isolated by enzymatic digestion, using previously described protocols with modifications (PMID: 31829938, PMID: 28830992, PMID: 26067211). Briefly, placental tissues were homogenized using a gentleMACS Dissociator (Miltenyi Biotec, San Diego, CA) either in an enzyme cocktail from the Umbilical Cord Dissociation Kit (Miltenyi Biotec) or in collagenase A (Sigma Aldrich, St. Louis, MO). After homogenization, cells were washed with ice-cold 1X PBS and resuspended in 1X PBS for cell counting, which was performed using an automatic cell counter (Cellometer Auto 2000; Nexcelom Bioscience, Lawrence, MA). Lastly, dead cells were removed from the cell suspensions using the Dead Cell Removal Kit (Miltenyi Biotec) and cells were counted again using an automatic cell counter. Viable cells were then pooled in groups and used for single-cell RNAseq library construction using the Chromium™ Controller and Chromium™ Single Cell 3' version 3 kit (10x Genomics, Pleasanton, CA), following the manufacturer's instructions. Sequencing of the single-cell libraries was performed on an Illumina Nextseq 500 instrument.