title: GSM3082701: D0-NC; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA extraction were preformed by a ammonium acetate and alcohol precipitation precedures. 1*10^8 cells were homogenized in 15 mL 4 M guanidine thiocyanate with RNaseA, after incubated at 37 oC for 30 mins,15 mL 8 M ammonium acetate were added and mixing. To precipitate DNA, 15 mL isopanol were added and mixing vigorously, the DNA were hooked to a new tube and washed by 70% ethanol. DNA were dissolved in 3 mL TE and extacted by 2 round of choloform. Two-step PCR amplification were performed on genomic DNA using Hifi DNA polymerase (Vazyme). The first PCR using  Outer-F(CTTGGCTTTATATATCTTGTGGAAAGGAC)and Outer-R(GGAAAAAGGCGGAGCCAGTACACGAC) to primeramplification the region containing sgRNA cassette. The second PCR included amplification to attach Illumina adaptors and to barcode samples(F:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTgtggaaaggacgaaacaccg, R:CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCAGTACACGACATCACTTTCC).The samples were multiplexing and sequencing in a single HiSeq-X run.
attributes.tag: GEO Accession
attributes.value: GSM3082701