Preferential intergenic transcript retention in the sperm DRNA fractions may present functional importance but remain unknown, and their possible existence should not be ignored in future research. Open in a separate window Figure 6 Peak annotation on entire genome. DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation. animals in generations G1 to G3 were kindly provided by Dr. C. Gunes (Germany) and Dr. A. Londono (Paris) and animals JAX mice (Jax strain B6, 129S-terttm1Yjc/J, stock# 005423) were purchased from your Jackson laboratory, Bar Harbor (experimental results of the latter are not shown). 2.2. Sperm Preparation Mouse spermatozoa were collected from your cauda epididymis. Motile spermatozoa were washed twice in MEM buffer (1 mM Na pyruvate, 0.5 mM EDTA, 50 U/mL penicillin, 50 mg/mL streptomycin, and 0.1% BSA) by centrifugation. Sperm pellets were suspended in phosphate-buffered saline (PBS) and centrifuged again. The pellets were washed twice in 50 mM HEPES buffer pH 7.5, 10 mM NaCl, 5 mM Mg acetate, and 25% glycerol. Samples of human sperm of unknown donors were kindly provided by the fertility clinic (20 September 2011 committee number: 2011/10). 2.3. Gel Electrophoresis of the DNA/RNA Complex After overnight incubation at 56 C in Tris buffer 20 mM pH 8, EDTA 50 mM, with 0.5% SDS, 20 M dithiothreitol and 400 g/mL Proteinase K, the total nucleic acid fraction was precipitated with ethanol, re-dissolved, further fractionated on Zymo-SpinTM columns (ZYMO-RESEARCH CORP, Irvine, CA, USA) and digested with Msp1 for resection of the MSX-122 telomeric and subtelomeric sequences up to the first CCGG site. After electrophoresis on either 8% acrylamide or 1.5% agarose gels and transfer to a nitrocellulose membrane, DNA and RNA were revealed by hybridization with 32P-labelled oligonucleotide probes. 2.4. Preparation of DNA-Bound RNA Extraction of total nucleic acids after enzymatic removal of the proteins was followed by fractionation of DNA and RNA with the standard TRIzolTM protocol (Rio GRIA3 et al., 2010). Both fraction of nucleic acid aqueous phase (RNA) and chloroform-water interphase separately ethanol precipitated, again, once more fractionated through binding onto Zymo-SpinTM columns (ZYMO-RESEARCH CORP, Irvine, CA, USA) and elution columns or by chloroform extraction ( accessed on 2 March 2021, ZR-Duet ? DNA/RNA MiniPrep Plus catalog number D7003). The free RNA was separated, and the fraction that remains bound to the DNA further purified after DNase digestion and purification with Zymo-SpinTM columns (ZYMO-RESEARCH CORP, Irvine, CA, USA). 2.5. Enzymatic Assays Restriction cleavage: extracts were incubated overnight with the indicated restriction enzymes (Promega) in 50 L containing 1C5 g total DNA. For electrophoresis, 25 L samples were loaded per lane. For extensive hydrolysis, extracts (20 L) were incubated for 30 to 60 min with RNase 0.5 g/L (which remove all RNA molecules), RNaseH 1 u/L (which remove only RNA molecules hybridized with the DNA), and DNase 10 u/L (which remove all DNA fragments), MSX-122 all provided by Roche Life Science (DNase free RNase ref. 11119915001, RNaseA ref. 10109169001, RNase H ref. M0297S). 2.6. Sequence Establishment DNA-bound RNAs were prepared from epididymal sperm 10 to 20 106 spermatozoa/male and from the total testis (estimated 60% of diploid cells) of a 6 month-old and a 14 month-old male, MSX-122 and two sperm samples from MSX-122 a man at reproductive age, all showing identical electrophoretic profiles of the DNA/RNA complexes. Mouse spermatozoa were collected from the cauda epididymis and ejaculated human.