|
| Status |
Public on Apr 24, 2021 |
| Title |
PBS_mouse1_local (gDNA) |
| Sample type |
SRA |
| |
|
| Source name |
Tibialis anterior muscule
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Tibialis anterior muscule
aav-donor: PBS
aav ratio: n/a
injection route: local intramuscular TA
tn5 gsp: g12
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was extracted from euthanized mice. Genomic DNA was isolated using the DNeasy kit (Qiagen) according to the manufacturer’s protocol. Total RNA was isolated using QIAshredder and RNeasy Plus kits (Qiagen). First-strand cDNA synthesis was performed using 500 ng total RNA per sample using the SuperScript VILO Reverse Transcription Kit (Invitrogen) and second-strand synthesis was performed using Klenow fragment DNA polymerase (NEB).
Tagmentation of 200 ng genomic DNA or second-strand products was performed using a 1:40 dilution of assembled Tn5. To enrich the targeted sequence, first round PCR using a genome specific primer was used with a reverse primer specific for the i7 adapter sequence inserted by the transposon. Second round PCR using a barcode primer specific for the custom adapter sequence was used to add 6-nucleotide experimental barcodes and the Illumina i5 adapter was used with the Tn5-Universal reverse primer.
Tn5-based tagmentation with target enrichment.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
PBS_m1_local
|
| Data processing |
Trim. The reads were trimmed for sequencing adapters and low-quality bases using Trimmomatic (v0.33).
Alignment and deduplication. Alignment of the trimmed reads was done using bwa-mem (v0.7.12) to the reference genomes (gDNA aligned to mouse genome GRCm38 + human DMD; cDNA aligned to human dystrophin cDNA). Aligned reads were tagged with their UMI using fgbio (v0.8.1) AnnotateBamWithUmis, and PCR duplicates were marked using Picard (v2.14.0) MarkDuplicates based on the UMI tag.
Alignment to reference bin sequences. Reference amplicons were built to align to the targeted locus and expected edits. To remove reads that are due to false priming, reads that do not contain the 20 bases directly adjacent to the GSP expected sequence were filtered out. To remove reads that do not extend far enough past the edit site, reads that are shorter than the required minimum length for binning were filtered out. On-site deduplicated reads were aligned to reference amplicons using bwa-mem (v0.7.12). Aligned reads were analyzed and binned based on editing outcomes using a custom-built script using python3 (https://github.com/siyansusan/DMD_integration_analysis.git). Reads that contain an indel ±15bp at the expected cute site or junction were identified. The number of distinct UMIs were counted for each edit to quantify frequency of editing events.
Genome_build: GRCm38 + human DMD
Supplementary_files_format_and_content: BAM file (curated and represents the collection of usable reads) for each sample. Manuscript methods include a link to github for access to analysis pipeline scripts; GEO users may find it useful to use the bam files to repeat analysis methods.
Supplementary_files_format_and_content: Matrix table with bin counts for every editing outcome and every sample
|
| |
|
| Submission date |
Apr 23, 2021 |
| Last update date |
Apr 25, 2021 |
| Contact name |
Adrian Oliver |
| E-mail(s) |
adrian.pickar@duke.edu, adrian.pickar@gmail.com
|
| Phone |
3016066963
|
| Organization name |
Duke University
|
| Street address |
101 Science Drive, CIEMAS Rm. 2323
|
| City |
Durham |
| State/province |
North Carolina |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE173224 |
Full-length dystrophin restoration via targeted genomic integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy |
|
| Relations |
| BioSample |
SAMN18849748 |
| SRA |
SRX10666407 |
