GSM5005815: V24Am; Mus musculus; RNA-Seq
For mouse samples, oviducts were collected 24 hrs after Veh or E2 treatment (n=5-6 mice/group) or at estrus (n=5 mice). 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. For human sample, To obtain cell singlets, the Fallopian tubes were transected so that the luminal portion of the tube was exposed. The mucosal lining of the tubes was gently dissected with a scalpel and enzymatically digested in a solution of 0.05% Trypsin (Sigma, T1426) dissolved in HBSS (Gibco) for 30 minutes, followed by incubation in Collagenase (5 mg/ml, Sigma C5138) and DNAse I (200 µg/ml, Sigma DN25) solution for 20 minutes. The cells were filtered through a 40 µm filter, collected by centrifugation and resuspended in DMEM/F12 (Gibco, 11-330-057) supplemented with 10% FBS (Sigma, F2442) and antibiotics (Gibco, 15-240-096). Live cells were sorted on a BD FACS Aria II at the Cytometry and Cell Sorting Core Laboratory at Baylor College of Medicine (BCM) and submitted for single cell sequencing at the Single Cell Genomics Core Laboratory at BCM. For mouse samples, scRNA-seq libraries were performed using manufacturer's protocol (10X Genomics Inc, Pleasanton, CA). Single Cell 3' v2 chemistry was used. Briefly, individual cells (~8,000 cells/run) were separated into droplets by Gel bead in EMulsion (GEM) technology using 10X Chromium Controller. Emulsion beads were broken and barcoded cDNAs were pooled for amplification. For human sample, scRNA-seq libraries were also performed using 10X Chromium Controller with Single Cell 3' v3 chemistry. For mouse samples, libraries generated were sequenced using an Illumina HiSeq4000, targeting 400M reads/run, paired-end, and 100bp read length. For human sample, libraries generated were sequenced using an Illumina NovaSeq 6000, targeting 300M reads/run, paired-end, 50bp read length.