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| Status |
Public on Mar 11, 2024 |
| Title |
R1-NHP-02-N-H3K9me3 |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Macaca mulatta |
| Characteristics |
tissue: liver
treatment: H3K9me3
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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.
Nuclei were mixed with activated Concanavalin A beads. After successive incubations with primary antibody (overnight) and secondary antibody (for 30 min) in antibody and Digitonin150 buffer respectively, the beads were washed with Digitonin150 buffer and resuspended in Digitonin300 buffer with 2.5uL of pA(G)-Tn5 for 1 hr and washed with the same buffer. Incubations were performed at room temperature in low-retention PCR strip tubes (Epicypher). Tagmentation was performed for 1 hr at 37C in Tagmentation buffer that provides MgCl2. Beads were washed with TAPS buffer and DNA material was released by adding SDS Release Buffer and incubating at 58C for 1hr. Quenching of SDS was performed by adding SDS Quench buffer and PCR was performed directly on this material. Universal P5 and indexed P7 primer solutions were used (see Supplemental Table 3 for sequences), and 17 cycles of PCR were performed. Clean-up was performed with AMPure beads, eluted in 15uL 0.1x TE buffer. Qubit and Bioanalyzer were used to verify library qualities before pooling samples for sequencing. The barcoded libraries were mixed to achieve equimolar representation aiming for a 10nM final concentration.
Cut & Tag. Sequencing of the same library was performed twice (R1, R2), once with Illumina HiSeq 4000 and once with Illumina NovaSeq SP100, 150 cycles total per lane, 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 HiSeq 4000 |
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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 mm10 build of the mouse 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 |
GPL23949 |
| Series (2) |
| GSE261044 |
Correlation of antigen expression with epigenetic modifications after rAAV delivery of a hyperactive human Factor IX variant in mice and rhesus macaques (CnT NHP) |
| GSE261047 |
Correlation of antigen expression with epigenetic modifications after rAAV delivery of a hyperactive human Factor IX variant in mice and rhesus macaques |
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| Relations |
| BioSample |
SAMN40287266 |
| SRA |
SRX23858774 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8133044_R1-NHP-02-N-H3K9me3_S33_L001.sort.rmdup.SeqDepthNorm.bedgraph.gz |
36.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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