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| Status |
Public on Jun 17, 2022 |
| Title |
LibB(gRNA11205)_spCas9 |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: human embryonic kidney cells
treatment: spCas9
molecule type: synthetic construct
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| Extracted molecule |
other |
| Extraction protocol |
The on-target and off-target sites were amplified by PCRs
Library cloning started by PCR amplification of the 170-nt oligonucleotide pool, Firstly, the CRISPR SURRO-seq oligos diluted to 1 ng/μl and then performed PCR amplifications using the primers: TRAP-oligo (BsmBI GGA)-F and TRAP-oligo (BsmBI GGA)-R. The PCR reaction was carried out using PrimeSTAR HS DNA Polymerase (Takara, Japan) following the manufacturer`s instruction. The PCR products of SURRO-seq oligos were then used for Golden Gate Assembly (GGA) to generate the plasmids library. 36 parallel GGA reactions were performed and the ligation products were pooled into one tube. Transformation was then carried out using chemically competent DH5αcells. For each reaction, 10μl GGA ligation product was transformed in to 50 μl competent cells and all the transformed cells were spread on one LB plate (15 cm dish in diameter) with Xgal, IPTG and Amp selection. High ligation efficiency was determined by the presence of very few blue colonies. To ensure that there is sufficient coverage of each TRAP in the 12K CRISPRTRAP-seq library, 42 parallel transformations were performed, and all the bacterial colonies were scraped off and pooled together for plasmids midi-prep (PureLinkTM HiPure Plasmid DNA Midiprep Kit). For NGS-based quality quantification of TRAP coverage, midi-prep plasmids were used as DNA templates for TRAP PCR amplifications, followed by gel purification and NGS sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
DNBSEQ-G400 |
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| Data processing |
FastaQC-v0.11.3(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp-v0.19.6(https://github.com/OpenGene/fastp) with default options were used for data quality control and filtering with the default parameters
BWA-MEM-v0.7.17 with default options was used to map the assembled data to the designed oligos sequence to preliminarily distinguish the data of each TRAP site
Julia-1.7.1 was used to calculate the read counts and editing read numbers to calculate the editing efficiency, R-4.1.3 was used to filter the editing efficiency.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include editing efficiency for each off-target site
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| Submission date |
Jun 17, 2022 |
| Last update date |
Jun 26, 2022 |
| Contact name |
Lin Lin |
| E-mail(s) |
lin.lin@biomed.au.dk
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| Organization name |
Aarhus University
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| Department |
Department of Biomedicine
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| Street address |
Høegh-Guldbergs Gade 10
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| City |
Aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
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| Platform ID |
GPL28038 |
| Series (1) |
| GSE206347 |
Massively targeted evaluation of therapeutic CRISPR off-targets in cells |
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| Relations |
| BioSample |
SAMN29177436 |
| SRA |
SRX15780378 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6251738_LibB_DMD_gRNA11205_spCas9.txt.gz |
646 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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