title: 10X Chromium Single-cell RNA sequencing of the female reproductive tract during aging in C57BL/6 mice
design: 10X Chromium Single-cell RNA sequencing of the female reproductive tract during aging in C57BL/6 mice
library_name: HA15M04_ut-01_p
library_construction_protocol: To generate single cell suspensions, freshly isolated whole tissues of ovary, oviduct and tissue pieces from uterus were treated by enzymatic digestion. All tissues were initially incubated separately in 2 ml Eppendorf tubes containing 0,25% trypsin in PBS and digested for 30 min at 37°C with gentle rocking. A second digestion buffer containing 25 mg/ml Collagenase I, 25 mg/ml Collagenase II, 50mg/ml Collagenase IV, and 25 mg/ml Dispase II in PBS was added, and tissues further dissociated for 2.5 hours at 37 °C with gentle rocking. Enzymatic digestion was quenched by addition of 10% FCS in PBS on ice. The cell suspensions were passaged through a 40µm cell strainer (Greiner Bio One) to remove cell debris and buffer residue. Cell suspensions were gently centrifuged at 350g for 7 min at 4°C. Cells were resuspended in PBS containing 0,04% FCS, 2 mM EDTA and propidium iodide (PI) was added to final concentration of 1 µg/ml prior to FACS sorting. 10x 3'-scRNA-seq ver. 3.1 (10X Genomics® User guide Rev.C and D) 10x 3'-scRNA-seq ver. 3.1 (10X Genomics® User guide Rev.C and D)