GSM6304124: ruptured tubal ectopic pregnancy 02 [REP2]; Homo sapiens; RNA-Seq
GSM6304124
The tissues of maternal-fetal interface were isolated and obtained for single cell sequence, with a proportion of 1 to 2 (maternal to fetus). Freshly collected tissues were flushed with ice-cold PBS. In details, for decidua and fallopian tube were digested with 20ml type I collagenase at 1.0 mg/ml in RPIM 1640 medium/10% fetal bovine serum (FBS) with gentle shaking at 37 °C for 1h and 20ml type I DNase at 0.1mg/ml for 5 mins in a shaking incubator. For placenta were digested twice for 30 min each at 37 °C with 30 ml mix digestive enzyme (15ml TrypLE and 15ml ACCUMAX) with gentle shaking. The digested suspension was filtered through a 100-µm cell strainer and 40-µm cell strainer, then the enzymatic reaction was stopped by adding 10% FBS. If the red cells were visualized, the RBC lysis step was performed: 1-2ml of RBC lysis buffer were added and triturated several times, then incubated for 1-2 minutes on ice. Cells were analyzed with a hemocytometer using trypan blue, and which survival rate is generally above 90%. This process was performed by Genergy Bio-Technology Corporation and in accordance with the manufacturer's protocol. Cells were counted and loaded onto the Chromium Controller (10X Genomics) for a target recovery of 8000 single cells. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were performed. Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina Nova6000 platform using 150-base-pair paired-end reads.