Genome-seq of Mus musculus: mESC (clone AN3-12)
AN3-12 mouse embryonic stem cells were lysed in Lysis buffer (10 mM Tris, pH 7.5; 10 mM EDTA, pH 8; 10 mM NaCl; 0.5% N-Laurosylsarcosine) and incubated at 60C overnight. DNA was ethanol precipitated using 0.2 M NaCl and the DNA was resuspended in 1x TE. Isolated gDNA was phenol-chloroform purified followed by ethanol precipitation. 2 g of purified gDNA was sheared in 130 l of 1xTE in a microTUBE AFA Fiber Crimp-Cap (6 x 16 mm) using the E220 Focused-ultrasonicator (Covaris) with the following settings: 140 W peak incident power, 10% Duty Factor, 200 cycles per burst, 80 seconds treatment time. The sheared DNA was bead-purified using Agencourt AMPure XP beads (Beckman Coulter) to select fragments between 250-500 nt. DNA library preparation was performed using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) and the library was sequenced in the paired-end 50 mode on a HiSeq 2500 instrument (Illumina).