GSM8007835: Proximal, Group 3, scRNAseq; Mus musculus; RNA-Seq
Each uterine tube was placed in sterile PBS containing 100 IU ml-1 of penicillin and 100 µg ml-1 streptomycin (Corning, 30-002-Cl), and separated in distal and proximal regions. Tissues from the same region were combined in a 40 µl drop of the same PBS solution, cut open lengthwise, and minced into 1.5-2.5 mm pieces with 25G needles. Minced tissues were transferred with help of a sterile wide bore 200 µl pipette tip into a 1.8 ml cryo vial containing 1.2 ml A-mTE-D1 (300 IU ml-1 collagenase IV mixed with 100 IU ml-1 hyaluronidase; Stem Cell Technologies, 07912, in DMEM Ham's F12, Hyclone, SH30023.FS). Tissues were incubated with loose cap for 1 h at 37°C in a 5% CO2 incubator. During the incubation tubes were taken out 4 times and tissues suspended with a wide bore 200 µl pipette tip. At the end of incubation, the tissue-cell suspension from each tube was transferred into 1 ml TrypLE (Invitrogen, 12604013) pre-warmed to 37°C, suspended 70 times with a 1000 µl pipette tip, 5 ml A-SM [DMEM Ham's F12 containing 2% fetal bovine serum (FBS)] were added to the mix, and TE cells were pelleted by centrifugation 300x g for 10 minutes at 25°C. Pellets were then suspended with 1 ml pre-warmed to 37°C A-mTE-D2 (7 mg ml-1 Dispase II, Worthington NPRO2, and 10 µg ml-1 Deoxyribonuclease I, Stem Cell Technologies, 07900), and mixed 70 times with a 1000 µl pipette tip. 5 ml A-mTE-D2 was added and samples were passed through a 40 µm cell strainer, and pelleted by centrifugation at 300x g for 7 minutes at +4°C. Pellets were suspended in 100 µl microbeads per 107 total cells or fewer, and dead cells were removed with the Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101) according to the manufacturer's protocol. Pelleted live cell fractions were collected in 1.5 ml low binding centrifuge tubes, kept on ice, and suspended in ice cold 50 µl A-Ri-Buffer (5% FBS, 1% GlutaMAX-I, Invitrogen, 35050-079, 9 µM Y-27632, Millipore, 688000, and 100 IU ml-1 penicillin 100 μg ml-1 streptomycin in DMEM Ham's F12). Cell aliquots were stained with trypan blue for live and dead cell calculation. Live cell preparations with a target cell recovery of 5,000-6,000 were loaded on Chromium controller (10X Genomics, Single Cell 3' v2 chemistry) to perform single cell partitioning and barcoding using the microfluidic platform device. After preparation of barcoded, next-generation sequencing cDNA libraries samples were sequenced on Illumina NextSeq500 System. The libraries were prepared with the chromium Single Cell 3' reagent kit (10X Genomics, Single Cell 3' v2 chemistry) following the maunfacturer's instructions. Live cell preparations were loaded with a target cell recovery of 5,000-6,000.