GSM6398388: Ovary, estrus; Bos grunniens; RNA-Seq
The pieces of ovarian tissue were washed in cooled RPMI 1640 medium containing 0.04% bovine serum albumin twice. The ovarian samples were further minced into approximate 0.5 mm3 pieces with a sterile scalpel blade in RPMI 1640 medium containing 0.04% BSA. The pieces were incubated with 1 mg/ml collagenase Type II in 0.25% trypsin-EDTA on ice overnight. The digestion was stopped by adding 10% of fetal calf serum. Next, the dissociated cells were collected by centrifugation at 160 × g for 3 min and incubated with advanced DMEM/F12 Glutamax containing 1% insulin-transferrin-selenium , 1% penicillin-streptomycin, and 27 IU/ml RNase-free DNase I at 37 °C for 1 h. The ovarian cells were resuspended in DPBS containing 2% FBS and passed through a 40 μm cell strainer. The cell suspensions containing high viability cells were prepared immediately. Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Chromium, PN-1000128) was used for gel beads in emulsion (GEM) formation. The cell suspensions, gel beads, and partitioning oil were loaded into a Chromium Single Cell Controller to produce single-cell GEM. Then, a primer containing Illumina TruSeq Read 1, 10X Barcode, and unique molecular identifier (UMI) was applied to produce barcoded full-length cDNA by reverse transcriptase reaction. The GEMs were broken and the pooled fractions were recovered. The first-strand cDNAs were purified with silane magnetic beads and amplified to generate sufficient mass for library construction. Enzymatic fragmention and size selection were used to optimize cDNA amplicon size. P5, P7, a sample index, and a TruSeq Read 2 were added to the amplicon via end repair, A-tailing, adaptor ligation, and PCR.