GSM7157529: Undifferentiated Spermatogonia Dnmt3a f/+ ; Dnmt3b f/- ; Stra8-iCre tg rep 1; Mus musculus; Bisulfite-Seq
For sperm cell sorting, mouse cauda epididymides were dissected into a Petri dish and fat patches were removed with forceps and scissors. Each epididymis was punctured with a needle and carefully squeezed into 100 ul of PBS with the help of two forceps. Sperm was transferred into an Eppendorf tube and was allowed to liquefy at room temperature for 5 minutes. To break sperm tails, cells were briefly sonicated with a Brandson Tip digital sonicator with 10% amplitude and 3 cycles of 0.5 sec ON / 2 sec OFF. Cells were stained with 2 ul/ml Hoechst 33342 (H3570, ThermoFisher) for 1h at 25°C with constant shaking and protected from light. Sperm cells were filtered through a 40 μm Nylon filter into 5 ml polypropylene tubes. Sperm cells were sorted for 1C DNA content. Pellets of 1 million sperm cells were incubated in 200μl of DNA extraction buffer (20mM Tris-HCl pH 7.5, 300mM NaCl, 2% SDS, 10mM EDTA, 200 μg/ml Proteinase K) for 16 hours at 55oC. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution was added to the samples. After a vigorous vortexing for 30 seconds and centrifugation at 16.000g for 10 minutes, the upper aqueous phase was transferred to new tubes. 0.05 μg/μl glycogen, 1/10 volume of 3M sodium acetate and 2.5 volumes of 100% ethanol were added, and the samples were incubated fot 2 hours at -80oC. DNA precipitated by centrifugation at 16000g for 30m minutes at 4oC. DNA pellet was washed once with 70% ethanol and resuspended in 20 μl. DNA was further purified using 2 volumes of Ampure XP beads (Beckman Coulter). 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.DNA was extracted from 10000 undifferentiated or differentiated spermatogonia cell samples by QIAamp DNA Micro Kit (Qiagen # 56304) according to the manufacturer's instructions. DNA was digested with 0.9μl MspI Fast Digest (Thermo Scientific FD0544) in 1X Tango buffer in a volume of 18 μl for 6 hours at 37oC. DNA ends were repaired by adding 1 μl Klenow Fragment, exo– (Thermo Scientific EP0421) and 1µl of nucleotide end-repair mix (1mM dATP; 0.1mM dGTP; 0.1mM dCTP prepared in 2X Tango buffer) and incubated for 45min at 37ᵒC, followed by enzyme heat inactivation at 75ᵒC for 15min. Then 6.25nM of 5mC sequencing adapter (NEB E7535S) was ligated with 1 μl T4 DNA Ligase, HC (Thermo Scientific EL0013) and 0.5 μl ATP solution (Thermo Scientific R0441) for 16 hours at room temperature. The looped adaptor ligated DNA fragments were subjected to USER (NEB M5508) digestion at 37 ᵒC for 20 minutes and used directly for bisulfite treatment using the Imprint DNA Modification Kit (Sigma MOD50) according to the manufacturer's 2-step modification procedure. The modified DNA was amplified with 13 cycles of PCR using Kapa U+ polymerase (Roche 07959052001) and indexed primers (NEB) and purified with 1.8X Ampure XP beads.