GSM8831107: Her2-GFP Screen Bulk; Homo sapiens; OTHER
To prepare genomic DNA, sorted cells were mixed with 5 x 106 non-infected RTK-FP cells to increase recovery, and the sorted and unsorted cell pellets were resuspended in 100 mL of TE buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA) per 5 x 106 cells. 900 mL of Lysis Buffer containing 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS and 0.2 mg/mL Proteinase K (Thermo Fisher Scientific) was added to 100 mL of cells. Cell lysates were incubated at 56°C and vortexed vigorously every 15 min, followed by an additional overnight incubation. After the incubation, 5M NaCl was added to lysates to yield final concentration of 0.2M NaCl. A phenol chloroform extractions was performed using MaXtract tubes (Qiagen) by mixing lysates with an equal volume of Phenol:Chloroform:Isoamyl alcohol 25:24:1 (Life Technologies), followed by centrifugation at 1500 x g and extraction by an equal volume of chloroform. The aqueous phase was treated with 25 mg/mL of RNAse A (PureLink, Life Technologies) for 1 hr at 37°C with periodic mixing followed by a second phenol chloroform extraction. The aqueous phase was combined 1:30 v/v with 3M Sodium acetate, pH 5.2 and the DNA was precipitated with 2.5 volumes of ethanol for 1 hr at room temperature. Precipitated DNA was pelleted by centrifugation at 4,000 x g for 1 hr at 4°C in a swinging bucket centrifuge. The DNA pellet was washed 3 times with 75% ethanol, allowed to dry and resuspended in 200 mL of water per 5 x 106 lysed cells. DNA was further solubilized by incubation for 1 hr at 55°C followed by passage 10-20 times through a 28-gauge needle. DNA purity was assessed using a NanoDrop spectrophotometer and its concentration was determined using Qubit Fluorometric Quantification (Thermo Fisher Scientific). Genomic sgRNA libraries were prepared by PCR amplification of all DNA extracted from sorted cells and 70 mg of DNA extracted from the unsorted control samples using common primers. 4 ml of the PCR products was used as a template for another round of PCR with barcoded primers. The products were purified using SPRIselect beads (Beckman Coulter), and their purity and concentration was determined using the 2100 Bioanalyzer instrument (Agilent)