GSM5599220: Normal ovarian tissue1; Homo sapiens; RNA-Seq
The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. Tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with 125 U/mL collagenase IV (Sigma), 25 U/mL collagenase I (Sigma) and 25 U/mL DNase I (Worthington) for 30 min at 37°C, with agitation. After digestion, samples were sieved through a 40µm cell strainer, and then centrifuged at 300g for 5 min. The supernatant was removed and the pelleted cells were suspended in red blood cell lysis buffer (Solarbio) to lyse red blood cells. The cells were re-suspended in RPMI-1640 medium containing 10% FBS and re-filtered through a 35μm cell strainer. Dissociated single cells were then stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer. The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec). RNA libraries were generated by 10xGenomics Single Cell 3' V3.1 Library Gel Bead Kit and prepared for sequencing using standard Illumina protocols