GSM2417782: Mixed Early rep2; Caenorhabditis elegans; OTHER
Samples were resuspended in 480 µl Lysis buffer (50 mM Tris-HCL, pH 8.0, 50mM EDTA, 100 mM NaCl, 1.5% Sarkosyl, 1% SDS) and incubated with 200 µg proteinase K at 42 degree overnight. Digested proteins and peptides were precipitated by addition of 200 µl 5M KOAc and centrifugation at 16,000g for 30 minutes at 4˚C. Genomic DNA was precipitated by adding 500 µl isopropanol and spinning at 16,000 g at 4˚C for 10 minutes. Genomic DNA pellets were washed with 70% ethanol and then resuspended in 300 µl STE (10mM Tris-HCL, PH8.0, 1 mM EDTA, 100mM NaCl). Residual RNA was digested by addition of 5U RiboShredder RNase Blend (Epicentre) at 37˚C overnight. Genomic DNA was precipitated with 30 µl 3M NaOAc and 1.7 ml ethanol pelleted at 10,000g for 10 minutes at 4˚C. DNA pellets were washed with 70% ethanol and resuspended in 30 µl TE (10mM Tris-HCl pH 7.5, 0.1 mM EDTA) and stored over night at 4˚C to allow complete resuspension of genomic DNA. Samples were stored at -80˚C. Three hundred ng purified Okazaki fragments were used to generate sequencing library similar to the previously optimized protocol . Following fragment ligation the total reaction was loaded on a 2% agarose gel; fragments corresponding to ~200-700 bp were purified from the gel using QiAquick gel extraction kit (Qiagen). Purified ligated fragments were amplified by (PCR 16) cycles using custom Illumina or Ion Torrent sequencing oligos
GEO Accession
GSM2417782