title: GSM4460457: OriSSDS Mouse Whole Testis Sample 4; Mus musculus; OTHER
library_construction_protocol: Mice were euthanized and testes were retrieved. The tunica albuginea was removed and both testes were suspended in 10 ml of DNAzol (Thermo-Fisher catalog number 10503027) and transferred to a Dounce homogenizer. Tissue and cells were disrupted with 5-7 strokes of a loose-fitting pestle. The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h. DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. To match conditions used for the RNA hydrolysis control, samples were incubated for 30 min at 37°C in Tris Buffer pH=8. Then, samples were heat denatured and spun through a Chromaspin TE-1000 size exclusion column (Clontech). SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns