GSM7157173: Differentiated Spermatogonia Dnmt3a f/+ ; Dnmt3b f/+  rep 2; Mus musculus; RNA-Seq
For spermatogonia, testicular cell suspension was prepared by incubation of seminiferous tubules in 200 U/ml Collagenase type I (Worthington Biochemical LS004196), 5 μg/ml DNAse I (Roche 10104159001), and 0.05%Trypsin (Gibco 25200056) in GBSS (Sigma G9779) as described in (60). Cells were stained with 1/200 anti-CD117/c-kit PE (eBioscience 12-1171-83), anti-CD324/E-Cadherin eFluor 660 (eBioscience 50-3249-82) and anti-CD49f/Integrin alpha 6 PE-Cyanine7 (eBioscience 25-0459-82) for 1 hour at 32oC with constant shaking and protected from light. After washing cells were incubated with 20 μg/ml with Hoechst 33342 (Thermo Fischer Scientific H3570) for 1 hour at 32oC with constant shaking and protected from light and then with 30nM DRAQ7 (Biostatus DR71000) for 5 minutes on bench. The stained cells were strained through a 40 μm nylon filter into 5 ml polypropylene tubes and were sorted on a BD FACSAria III cell sorter fitted with a 70 μm nozzle (Becton Dickinson) using a 375nm laser to excite Hoechst 33342, a 561nm laser to excite PE and PE-Cy7 and a 633nm laser to excite DRAQ7 and eFluor600. Cells were first gated for FSC and SSC to exclude debris. Then the live cells were selected based on the absence of DRAQ7 signal detected using 755LP, 780/60BP. Then we gated for cells positive for Hoechst 33342 emission which was detected using a 670LP (Hoechst-Red) and 450/20 BP (Hoechst-Blue). We gated for the Hoechst-Redlow and Hoechst-Bluemid population which is enriched for 2N spermatogonia. Fluorescence for PE was detected using a 582/15BP filter, for PE-Cy7 was detected using 735LP,780/60BP filter and for eFluor660 using a 660/20BP filter. Undifferentiated spermatogonia were sorted as CD324high, CD49fhigh, CD117low and differentiated spermatogonia were sorted as CD324low, CD49flow, CD117high. RNA from Spermatogonia (10.000 per sample) was extracted with Single Cell RNA Purification Kit (Norgen, 51800) . Ovation® SoLo RNA-Seq Library Preparation Kit (Nugen, 0501-32)