GSM8339548: AEb14-2_157_CTR_M; Homo sapiens; RNA-Seq
Placental chorionic villi were separated from fetal membranes and decidua, washed in DPBS wash, and mechanically homogenized. Placental tissue was then serially digested in Collagenase Digestion Buffer, Trypsin Digestion Buffer, and Collagenase Digestion Buffer 2. Undigested tissue was removed by passage through sterile gauze and 100uM filter. The cell suspension was centrifuged at 257g for 8 min at 4°C, washed, spun again, and resuspended in media.  The cells were then suspended in 4mL of 20% Percoll and 5mL of 35% Percoll was underlayed through a #1 glass Pasteur pipette. After centrifugation for 30 minutes at 4°C without brake at 1000g, cells were isolated from the interphase layer, washed in media, and spun at 257g for 8 minutes at 4°C. Cell pellets were immunopurified by negative selection by incubation with anti-EGFR (to remove syncytiotrophoblasts) and anti-CD10 (to remove fibroblasts) conjugated to magnetic Dynabeads, prepared as previously described, for 20 minutes at 4°C. Tubes were placed on a DynaMagTM magnet for 5 minutes to magnetically bind cytotrophoblasts Cryopreserved HBCs were thawed at 37°C and diluted with RPMI 1640 including 10% FBS and 1% Pen/Strep. The cell suspension was centrifuged at 300g for 5 min at room temperature, with the brake off. The supernatant was aspirated and the cell pellet was resuspended in media.  Dead cells were removed using OptiPrepTM Density Gradient Medium (Sigma) and cell count and viability of cells were calculated using LunaFX7 automated counter. 10x Genomics platform with a loading target of approximately 10,000 viable cells/sample.  Libraries were sequenced on an Illumina NextSeq 2000 P3 flowcell machine with a sequencing target of 25,000 reads per cell.