title: GSM4305600: SRE_Elk_Input_R2; Mus musculus; OTHER
library_construction_protocol: 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 16000 xg 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).
attributes.tag: GEO Accession
attributes.value: GSM4305600