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| Status |
Public on Jun 10, 2016 |
| Title |
1a-lo-5-P90_S1_L001_R1_001_OUTPUT_AF_SOL_580.fasta |
| Sample type |
SRA |
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| Source name |
type I unc-22 RVL library
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| Organism |
synthetic construct |
| Characteristics |
target random variant library (rvl): type I unc-22 RVL library
molecule subtype: library DNA
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| Treatment protocol |
Cas9 in vitro cleavage specificity assay
Cas9 in vitro cleavage assays and gRNA transcription (17, 18, and 20 nt complementarity) were performed as described in Fu et al. 2014 (doi: 10.1093/nar/gku1102). Over 4000 unique species were assayed in vitro.
Cas9 in vivo cleavage specificity assays
Starting cultures were grown overnight at 30oC in synthetic complete media –Ura. This culture was then used to inoculate experimental cultures to OD 0.1 in YP Galactose media with 250ng/ml anhydrotetracycline (ATC). To control for possible effects of non-homologous end joining (NHEJ) events, we did our initial experiments in both wild BY4741 strain and a KU70 null NHEJ deficient strain. 17, 18, 20 and 20+G nt complementarity gRNA strains were grown for 12 hours in inducing conditions. No detectible difference in the cleavage pattern was observed. Wild type BY4741 was used for all further experiments. Approximately 3000-4500 unique species were assayed in vivo.
Further experiments were done with 18 and 20 nt complementarity versions of the gRNAs in BY4741. 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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
No processing. There is just a calculation of retention scores for each sequence with n>=50 counts in the control samples.
Computational Methods: Calculation of retention and normalization:
We use the log2 of retention in the PCR pool as a measure of target cleavage by Cas9, with a retention score calculated for each sequence in each experiment.
For each sequence 'X':
Retention[X]= log2(RepresentationX[Cleaved Library]/RepresentationX[Uncleaved Library])
Representationx in a library gives the ratio between:
[Instances of sequence X in the library]/[Instances of a reference population in the library]
As reference populations we used either an aggregate of all sequences with 4-7 mismatches to the original trigger, or (for the unc-22A library) an "internal control" population comprising a subset of plasmids from the protospacer 4 library. Comparable results were obtained with these two references for normalization.
Supplementary_files_format_and_content: Raw data is in fastq format. Data used to calculate retention is in fasta format. Refer to PMID: 25399416 for how to calcuate retention. This analysis step should be performed by further processing depending on user parameters.
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| Submission date |
Mar 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Fire |
| E-mail(s) |
xuhua@stanford.edu
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| Phone |
916-307-2869
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| Organization name |
Stanford
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| Department |
Genetics
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| Lab |
Andrew Fire
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| Street address |
300 Pasteur Drive Lane building L302
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL17769 |
| Series (1) |
| GSE79667 |
Cas9 target DNA specificity in vitro and in vivo (S. cerevisiae) |
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| Relations |
| BioSample |
SAMN04589019 |
| SRA |
SRX1667589 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2100670_1a-lo-5-P90_S1_L001_R1_001_OUTPUT_AF_SOL_580.fasta.gz |
9.0 Mb |
(ftp)(http) |
FASTA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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