GSM2100847: 3e-lo-10-gfp2.20nt_S20_L001_R1_001_AF_SOL_672_OUTPUT.fasta; synthetic construct; OTHER
Cas9 in vitro cleavage specificity assay In vitro Cas9 cleavage assays and gRNA transcription (17, 18, and 20 nt of complementarity) were performed as described in PMID: 25399416. Cas9 in vivo cleavage specificity assays Yeast culturing and sample collection was performed using a cell-screening platform that integrates temperature-controlled absorbance plate readers, plate coolers, and a liquid handling robot. Briefly, 700 ul yeast cultures were grown in 48 well plates at 30oC with orbital shaking in Infinite plate readers (Tecan). To maintain cultures in log phase over 10 doublings, 80 uls of the culture was removed when it reached an OD of 0.76, added to a well containing 620 ul of media, and then allowed to grow further. After two such dilutions, 600 uls of the culture was collected and saved to a 4oC cooling station (Torrey Pines) when it reached an OD of 0.76. This amounted to approximately 10 culture doublings from the beginning of the experiment. Pipetting events were triggered automatically by Pegasus Software and performed by a Freedom EVO workstation (Tecan). After sample collection, yeast plasmids were purified using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research).
GEO
GSM2100847