GSM4310157: Nanog_DoubleSorted_Top_R2; Mus musculus; OTHER
For the primary screen, genomic DNA was isolated from the frozen cell pellets by using the DNeasy Blood and Tissue kit (Qiagen) following the manufacturer's instructions. For the secondary screens, genomic DNA from frozen cell pellets was isolated via Phenol/Chloroform extraction due to higher yields. Briefly, cell pellets were thawed and resuspended in 250µl of Lysis buffer (1% SDS (Thermo Fisher Scientific), 0.2M NaCl and 5mM DTT (Roth) in TE Buffer) and incubated over night at 65°C. 200µg of RNAse A (Thermo Fisher Scientific) was added to the sample and incubated at 37°C for 1h. 100µg of Proteinase K (Sigma) were subsequently added followed by a 1h incubation at 50°C. Phenol/Chloroform/Isoamyl alcohol (Roth) was added to each sample in a 1:1 ratio, the mixture was vortexed at RT for 1 min and subsequently centrifuged at 16000 xg for 10 min at room temperature. The aqueous phase was transferred to a new tube, 1 ml 100% EtOH, 90 µl 5 M NaCl and 1µl Pellet Paint (Merck) was added to each sample, mixed, and incubated at -80°C for 1 hr. DNA was pelleted by centrifugation for 13000 rpm for 15 min at 4°C, pellets were washed twice with 70% EtOH, air-dried and resuspended in 50µl H2O. The PCR amplification of the sgRNA cassette was performed in two PCR steps as described previously with minor modifications (Shalem et al. 2014).