|
| Status |
Public on Sep 01, 2020 |
| Title |
15235X1 treatment with gRNA |
| Sample type |
SRA |
| |
|
| Source name |
AD-293
|
| Organism |
Homo sapiens |
| Characteristics |
group: SpCas9 witht gRNA.
|
| Treatment protocol |
To produce the gRNA, in vitro transcription was performed with MEGAshortscript™ T7 Transcription Kit (Thermofisher). SpCas9 protein was obtained from NEB (M0386M). The reaction was performed in 8µg genomic DNA (AD-293), 120pmol (300nM) SpCas9, 120pmol (300nM) or 360pmol (900nM) gRNA with 1X NEBuffer™ 3.1 (total volume is 400µL) at 37℃ for 8 hours. We found that 360pmol gRNA (Cas9 : gRNA = 1 : 3) showed efficient digestion at the target site. Therefore, we proceeded with 360pmol gRNA treated gDNA for deep sequencing.
|
| Growth protocol |
10%FBS/DMEM
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Quick-DNA Plus Kit (ZymoResearch)
Illumina TruSeq Nano DNA Sample Prep kit
NovaSeq 2 x 150 bp Sequencing 30X Human Whole Genome
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
In vitro SpCas9 digested genomic DNA
|
| Data processing |
The human GRCh38 FASTA file was downloaded from Ensembl and a reference database was created using bowtie2 version 2.3.4.
Adapters were trimmed out of reads using Cutadapt 1.16 and then aligned using Bowtie 2 in end-to-end mode (full options --end-to-end --sensitive --no-unal -k 20).
The aligned reads were loaded into R using the GenomicAlignments package and total coverage and read start coverage were calculated for the plus and minus strands.
Positions with 5 or more read starts were compared to the total coverage and read starts with less than 25% of total coverage were removed.
The filtered read starts on the positive and negative strands were joined to find predicted cut sites with either no overlap (blunt end), 1 base pair gap or 1 base pair overhang.
Genome_build: GRCh38
Supplementary_files_format_and_content: BigWig file from BamCoverage with binSize 1 and samFlagExclude 1792
|
| |
|
| Submission date |
Mar 15, 2020 |
| Last update date |
Sep 02, 2020 |
| Contact name |
Hironori Uehara |
| E-mail(s) |
uhironori_0916@yahoo.co.jp
|
| Organization name |
Loma Linda University
|
| Department |
Ophthalmology
|
| Lab |
Ambati lab
|
| Street address |
11021 Campus st
|
| City |
Loma Linda |
| State/province |
California |
| ZIP/Postal code |
92350 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE146997 |
Digenome for off-target analysis: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy |
| GSE146999 |
Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy |
|
| Relations |
| BioSample |
SAMN14380126 |
| SRA |
SRX7913592 |
