GSM8136427: Adjacent 2; Homo sapiens; RNA-Seq
Adjacent myometrium tissue of uterine niche (Adjacent1 & Adjacent2), well-healed cesarean scar tissue (Control) and uterine niche tissue (Niche1 & Niche2) were harvested. These tissues were cut into small pieces with scissors and then incubated in DMEM medium containing collagenase I (Gibco, 17100017) and collagenase II (Gibco, 17101015) at 37°C with gentle shaking for 40-60 minutes. The digested suspension was filtered through a 100-µm cell strainer (CSS-013-100, BIOFIL). Then, the enzymatic reaction was stopped by adding 10% fetal bovine serum (FBS). The cells were pelleted by centrifugation at 350 × g for 5 minutes at 4°C. Red blood cell lysis buffer (Beyotime Biotech, C3702) was used to remove red blood cells. After removing the supernatant, the cells were resuspended in 100 μL of DMEM medium. Cell analysis was conducted using a Trypan blue cell counter, and the cell viability was consistently above 80%. The cell concentration for loading onto the 10x Genomics Chromium™ machine ranged from 700 cells/μL to 1200 cells/μL. Cells were counted and loaded onto the Chromium Controller (10X Genomics) to recover a target of 8000 single cells. The process involved the generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification, and library construction. Library quantification was performed using Qubit before pooling. The final library pool was sequenced on an Illumina Nova6000 platform using 150-base-pair paired-end reads. Aligning to the GRCh38 human genome, Cell Ranger software (version 6.0.3) was used for quality control and read counting of Ensembl genes.