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| Status |
Public on Mar 11, 2024 |
| Title |
R1-NHP-42_KP1 |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Macaca mulatta |
| Characteristics |
tissue: liver
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| Treatment protocol |
Rhesus macaques were intravenously infused with rAAV packaged using different capsids. The rAAV vector expressed CpG depleted, codon-optimized huFIX under control of a liver-specific promoter.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Macaques were euthanized 3.5 months after rAAV injection and liver tissue from 4 lobes was flash-frozen. Frozen liver tissue was pulverized on dry ice and aliquoted. Equal amounts of pulverized liver tissue from each lobe were combined and ~100 mg of this pool were homogenized in RINO 1.5-ml Screw-Cap tubes filled with stainless steel beads and 1mL of NE buffer using a bead homogenizer (Next Advance Bullet Blender Storm - BBY24M) at speed 8 for 6 minutes. The homogenate was transferred into to a 50mL tube with 12mL of NE buffer and incubated on ice for 10 min. Nuclei were pelleted by a 5 min centrifugation at 600g, the supernatant was decanted, and the pellet was resuspended in 1mL of NE buffer. After passing nuclei through a 0.45um cell strainer quantitation was performed after Trypan Blue staining using an automated cell counter (Countess, Thermo Fisher) and diluted accordingly. 1e5 nuclei per target protein were used for Cut&Tag.
Total gDNA was extracted using a QIAamp DNA Mini kit (Qiagen) including RNAse treatment. The protocol for Illumina DNA prep was followed as per manufacturer’s instructions with 100ng of DNA as input, 30min tagmentation and 8 cycles of PCR. The same indexed primers as for Cut&Tag were used for PCR amplification.
Illumina DNA prep kit. Sequencing was performed with Illumina NovaSeq 6000, 150 cycles total per lane (2 lanes total), 2x75 paired-end reads, depth of >15M reads per sample.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were trimmed with Trimmomatic-0.39 program for classical Illumina adapters. Aligned using Bowtie2 version 2.2.5 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.5/) with options: --local --very-sensitive-local --nounal --no-mixed --no-discordant --phred33 -I 10 -X 700; mapping was performed using the Mmul_1 build of the rhesus macaque genome, merged with viral genome sequence (gAAV).
PCR duplicates were eliminated with “MarkDuplicates” command of Picard version 2.23 (https://broadinstitute.github.io/picard/index.html).
Tracks were made as bedgraph files of normalized counts, using deeptools 3.3 (https://deeptools.readthedocs.io/en/develop/content/list_of_tools.html) bamCoverage with options: --binSize 1, --normalizeUsing CPM; track plots were made with R package Gviz 4.1. Coverage was calculated as SUM[(normalized count*(end-start position+1))/gAAV size] for each dataset. Importantly, ITR regions were removed from the calculation, as their coverage is overrepresented in all samples, perhaps due to recognition of ITRs from ssDNA.
Assembly: Mmul_1
Supplementary files format and content: Bedgraph
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| Submission date |
Mar 06, 2024 |
| Last update date |
Mar 11, 2024 |
| Contact name |
Katja Pekrun |
| E-mail(s) |
kpekrun@stanford.edu
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| Organization name |
Stanford School of Medicine
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| Department |
Pediatrics & Genetics
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| Lab |
CCSR 2110
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| Street address |
269 Campus Drive
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| City |
STANFORD |
| State/province |
CA |
| ZIP/Postal code |
94025 |
| Country |
USA |
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| Platform ID |
GPL27943 |
| Series (2) |
| GSE261046 |
Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (Tn5 NHP) |
| GSE261047 |
Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques |
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| Relations |
| BioSample |
SAMN40287015 |
| SRA |
SRX23858304 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8133094_R1-NHP-42_KP1_N_S1_L001_R1_001.sort.rmdup.SeqDepthNorm.bedgraph.gz |
498.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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