title: GSM8701337: Ovatian Cortex Fresh-1; Homo sapiens; RNA-Seq
library_construction_protocol: For 10X Genomics strategy, the fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. Before FACS, the Blood cells were depleted by Red Blood cells lysis buffer (BD Pharmingen). Cells were then labeled with DAPI (4',6-diamidino-2-phenylindole, Cat number: D9452, Sigma, 1:1000), and DAPI- cells (live cells) were sorted through a MoFlo FACS machine (MoFlo Astrios EQ, Beckman Coulter) directly into PBS containing 0.04% BSA. For Smartseq2 strategy, fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. RPMI 1640 medium containing 10% FBS was used to stop digestion. The resulting cell suspension was then transferred into a 3.5 cm dish. Then, oocytes were manually picked under a dissection microscope and transferred to a PBS drop (containing 0.1% BSA) by mouth pipetting. Finally, the oocytes were placed separately into an individual PCR tube with lysis buffer and stored at -80°C for subsequent experiments. For 10X Genomics strategy based single-cell RNA sequencing, the single-cell RNA library was built via 10x Chromium platform, and FACS-enriched live ovarian cells were loaded per channel approximately. The libraries were built via the Chromium platform and Chromium Single Cell 3' v2 chemistry. Finally, the libraries were loaded on an illumine NovaSeq 6000 System (Illumina, San Diego) with a PE150 sequencing strategy. For Smart-seq2 strategy based single-cell RNA sequencing, scRNA-seq libraries were constructed according to the published protocol with modifications [20b]. Brieﬂy, the constructed libraries of each oocyte will undergo preliminary screening through the marker genes (DDX4 for oocyte and NR5A2 for granulosa cells) of oocytes and granulosa cells via qPCR, aiming to exclude multi-cell samples. Then, the libraries were sequenced on an Illumina NovaSeq 6000 platform with a 150-bp paired-end read length by Novogene.
identifiers.namespace: GEO
identifiers.id: GSM8701337_r1