Illumina NovaSeq 6000 sequencing; 10X Chromium Single-cell RNA sequencing of the female reproductive tract in young, pregnant and aged C57BL/6 mice
10X Chromium Single-cell RNA sequencing of the female reproductive tract in young, pregnant and aged C57BL/6 mice
To generate single cell suspensions, freshly isolated whole tissues including ovary, oviduct and tissue pieces from uterus, cervix, vagina and spleen 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 30min 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. 3.0 and 3.1 (10X Genomics® User guide Rev.C and D) 10x 3'-scRNA-seq ver. 3.0 and 3.1 (10X Genomics® User guide Rev.C and D)