title: GSM3381981: Brunello screen GFP+ (1); Homo sapiens; OTHER
library_construction_protocol: SOX17GFP+ and SOX17GFP- cells are sorted by fluorescence activated cell sorting. Genmoic DNA were extracted by using Qiagen Maxi Kit (Qiagen 13362) A two-step PCR method was performed to amplify the gRNA sequence for Hi-Seq. For the first step, 380 _g of DNA per sample (6.6 _g of genomic DNA per 1 million cells) was used to perform PCR using Herculase II fusion DNA polymerase (Agilent, 600679) in order to achieve a 1,000-fold converge of the GECKO library containing 58,028 gRNAs. In total, we performed 38 separate 100 _l reaction with 10 _g genomic DNA per sample for 18 cycles and combined the resulting amplicons. For the second step, 5 _l of the product from the first PCR was used in a 100 _l PCR reaction for 24 cycles with primers to attach Illumina adaptors for barcoding. Gel purified amplicons from the second PCR were quantified, mixed and sequenced using a HiSeq 2500 (Illumina) at MKSCC Integrated Genomics Operation (IGO).
identifiers.namespace: GEO
identifiers.id: GSM3381981