GSM5912296: High Old/new-rep1 [A01]; Homo sapiens; OTHER
Sorted cells were lysed in STE buffer (10mM Tris pH8.0, 100mM NaCl, 1mM EDTA, 0.4%SDS, 0.2mg/mL Proteinase K). 5µL RNaseA (2mg/mL) was added after Proteinase K reaction. Lysate were then separated by phenol-chloroform-isoamyl alcohol (PCI) and the aqueous layer was collected for ethanol precipitation. Precipitated DNA was resuspended in 30uL H2O. Unsorted cells were lysed in Tissue & Cell Lysis Solution (Lucigen) and the protein was removed by centrifuge after adding MPC Protein Precipitation Solution. The supernatant was collected, precipitated by isopropanol, and resuspended by 1.2mL H2O/3x10^7 cells. Guides were amplified from genomic DNA by ExTaq following the protocol from previously published work(Sanson et al., 2018). PCR product was purified by AMPure XP.