GSM7815935: oviduct, V2h_Isth; Mus musculus; RNA-Seq
Oviducts were pooled and collected in Leibovitz-15 media supplement with 1% fetal bovine serum (L15+1%FBS) and placed on ice. For Veh and E2-treated samples, the oviduct was dissected into distal (infundibulum with ampulla, referred to as InfAmp) or proximal (isthmus with UTJ, referred to as IsthUTJ) regions. To isolate oviductal cells, oviducts were quickly uncoiled by trimming the mesosalpinx in order to locate the ampulla-isthmus junction (AIJ). Then, the tissues were dissected at the AIJ region for subsequent InfAmp and IsthUTJ cell isolation. For estrus samples, the entire length of the oviduct was used. After dissection, 0.25% trypsin-EDTA was injected into the oviduct using a blunted 30-gauge needle until the trypsin was visibly flushed out from the other end of the oviduct. The tissues were then placed in a tube containing 500 µL of trypsin and incubated on ice for 30 min and at 37°C for another 30 min. After the incubation, 500 uL of FBS was added to stop the enzymatic activity of trypsin. Oviductal cells were then flushed out from oviductal tubes with 4°C L15+10%FBS. At this stage, the epithelial, stromal, and parts of muscle cell layers were visibly flushed out. Only translucent longitudinal muscle cells were left intact. Cell clumps and tissue debris were strained twice through 40-µm cell strainers. Cells were then spun down and resuspended in 0.04% bovine serum albumin in phosphate-buffered saline. The final cell concentration was targeted for 8,000 cells/run. 10x library was constructed according to manufacturer's protocol using Chromium Single Cell 3' v3 reagents. libraries generated were sequenced using an Illumina NovaSeq600, targeting 1 billion reads/lane, paired-end, and 100bp read length.