GSM8816187: U2OS_Brunello_1_high; Homo sapiens; OTHER
genomic DNA extraction - Cell pellets were resuspended in a minimum of 500 ul (per 5 x10^7 cells) of Digestion Buffer ( 10 mM Tris-HCl pH8.0, 150 mM NaCl, 10 mM EDTA, 0.1% SDS) supplemented with 200 ug/ml of proteinase K (Thermo EO0491) and 30 ug/ml of RNAase A (DNAase and RNAase Free, Thermo EN0531) and incubated at 37oC for 3 hrs and then 55oC overnight. Lysates were then mixed 1:1 with Phenol/chloroform/isoamyl alcohol (PCI) solution (25:24:1) pH 7.8-8.2 (100mM Tris) (Sigma 25666) and centrifuged at >16,000g for 10 mins at room temperature. The supernatant was transferred to a new tube and 0.3 vol of 24:1 chloroform/isoamyl alcohol solution (Sigma C0549) added to the phenol extracts and reextracted by vortexing and centrifuging.  The supernatants were combined and 1 ul of 15 mg/ml GlycoBlue added with 0.5 vol of 7.5 M ammonium acetate (final concentration of 2.5 M).  0.7 Vol of Isopropanol was then added, mixed by inversion, incubated at room temperature for 5 mins and centrifuged at 16,000g for 30 mins at 4 oC. Genomic DNA pellets were washed with 70% ethanol, centrifuged at >16,000g for 5 mins at 4 oC, air dried at 42oC, resuspended in 10 mM Tris pH 8.0 overnight at 42oC and sheered by 5x -80oC freeze thawing prior to PCR. For PCR amplification, gDNA was divided into 50 μL reactions such that each well had at most 5 μg of gDNA. 0.2  μl of AmpliTaq Gold (1U, 4311820, Thermo) was added per 50 ul reaction with 2 mM MgCl2 and 1 μl of 10 μM P5 F hU6 5' TCTTGTGGAAAGGACGAAACACCG 3' and P7 R sgRNA – 5' TCTACTATTCTTTCCCCTGCACTGT 3' primers in a PCR program of 95oC for 3 min, and 30 cycles of 94oC for 30 sec, 58oC for 30 sec, 72oC for 30 sec, followed by extension at 72oC for 10 mins. The PCR reactions from each condition were combined and purified by SPRI bead (Beckman Coulter B23317) purification, removing gDNA and primers. 10 ng of PCR product was then used in  the next set of 10 x PCRs using 15 cycles and staggered barcoding primers to add sequencing adaptors. The PCRs were then pooled and purified by SPRI bead purification, quantified and sequenced on an Illumina NextSeq500 1x75bp high output run at the South Australia Genome Centre (SAGC).