GSM5213636: OriSSDS Mouse ESCs Sample 2; Mus musculus; OTHER
Cells were suspended in 8 ml of DNAzol (Thermo-Fisher catalog number 10503027). 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. 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.