GSM9140737: adult testicular cells; Homo sapiens; RNA-Seq
All patients have provided written informed consent. Testicular tissues of patients with OA were minced with sterilized scissors after washing three times with PBS, then were digested in collagenase type and trypsin at 37 °C for 30 min. The digestion process was stopped with DMEM containing 10% FBS. After centrifugation and resuspension, single testicular cells were randomly selected into the prepared lysis buffer through a mouth pipette and stored at −80 °C for further analysis. Sorted single-cell or microregion tissue samples were removed from the −80 °C storage and thawed at room temperature for 10 min to facilitate sample lysis and RNA release, then transferred to a preheated PCR thermocycler, incubated for 3 min at 72 °C and immediately placed on ice afterwards. Next, 10 µl of RT-PCR mix containing 1.2 µL dNTP mix (10 mM), 0.252 µL RNase Inhibitor (40 U/µl), 0.1 µL DTT (100 mM), 0.328 µL MgCl2 (279 mM), 0.18 µL dCTP (100 mM), 2 µL Betaine (5 M), 0.368 µL CBTi-TSO (50 µM), 0.172 µL of Superscript Ⅳ / Maxima H (200 U/µL, ThermoFisher Scientific) and 5 µL of KAPA HiFi HotStart Ready Mix (2×, Roche) were added to synthesis full-length dsDNA. The following RT-PCR program was set to: 60 min at 50 °C, 98 °C for 3 min, then 20 cycles of (98 °C for 20 s, 67 °C for 20 s, 72 °C for 6 min). The cDNA concentration was quantified using the Qubit 4.0 fluorimeter, and size distribution was assessed using capillary electrophoresis on a Fragment Analyzer system (Agilent Bioanalyzer). 1 μl unpurified amplified cDNA working dilution (5 ng/µl) of each sample was transferred to a new plate before adding tagmentation reaction mix as described above. Of note, each sample used unique combination barcoding. Samples were incubated for 10 min at 55 °C. Then, all tagmented products were mixed into 1.5- or 2 ml PCR tube, purified with 1.5× AMPure XP beads for termination of the tagmented reaction and collection, followed by elution with 20 µl nuclease-free water.