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| Status |
Public on Aug 07, 2023 |
| Title |
Sperm_untreated_NanoNOME_Whole_genome |
| Sample type |
SRA |
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| Source name |
Sperm
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| Organism |
Mus musculus |
| Characteristics |
cell type: Sperm
genotype: WT
treatment: GpC methylase (NEB)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 2 million sperm were on-ice lysed with hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin) for 10 min. Sperm were pelleted down and resuspended in 500 ul of 1x GpC buffer containing 150 μl of 1 M sucrose, 1.5 μl of 32 mM S-adenosylmethionine (SAM; NEB, B9003) and 50 μl of M.CviPI (NEB, no. M0227L). The suspension was carefully mixed and incubated for 7.5 min at 37 °C, followed by a boost with an additional 25 μl of M.CviPI and 1.5 μl of SAM for 7.5 min. The reaction was stopped by the addition of 25 μl of 10% SDS, 5 μl of 0.5 M EDTA, 6 μl of 20mg/mL proteinase K, and 28 μl of 1M DTT followed by overnight incubation at 55 °C. The total sperm DNA was recovered by PCI and ethanol precipitation.
For whole-genome nanopore sequencing, 1 ug of sperm DNA was used for library preparation using Oxford Nanopore Ligation-based library prep kit (ONT, SQK-LSK110). For target-enriched sequencing, single-stranded CRISPR–Cas9 DNA oligonucleotides (20 nt) were designed using the ChopChop v.3 designer tool (chopchop.rc.fas.har- vard.edu) and purchased from IDT. sgRNA were prepared using EnGen@sgRNA synthesis kit (NEB, no. E3322S). RNP complexes were assembled individually by combining 8 μl of nuclease-free water, 1.5 μl of NEBuffer r3.1 (NEB), 3 μl of 300 nM sgRNA, 1 μl of Cas9 Nuclease (NEB, no. M0386), followed by a 20-min incubation at 25 °C. The different RNP complexes were then pooled in a single tube. Per experiment, 3-4 μg of sperm gDNA was dephosphorylated with 10 μl of rSAP (NEB, no. M0371S) and 16 μl of 10× rCutSmart buffer (NEB, no. B6004S) for 30 min at 37 °C, then heat inactivated at 65 °C for 5 min. DNA was then purified through PCI and ethanol precipitation. The dephosphorylated DNA sample was then combined with the pool of RNP complexes in a 1:9 ratio. The reaction was incubated for 30 min at 37 °C to enable Cas9 cleavage. After digestion, Cas9 was inactivated by the addition of a 1/25 volume of 20 mg/mL proteinase K followed by a 15-min incubation at 55 °C. Cleaved and purified DNA was purified by PCI and ethanol precipitation and was subjected to dA-Tailing using Klenow Fragment (NEB, no. E6053). Reactions were incubated for 30 min at 37 °C and immediately subjected to adapter ligation as per the manual of Oxford Nanopore Ligation-based library prep kit (ONT, SQK-LSK110).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
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| Data processing |
Base calling was performed on the raw FAST5 files with Guppy (v.3.0.3 or v.5.0.11, ONT), using a configuration file for high-accuracy DNA base calling on an R10.4.1 pore at 450 bases s–1. The resulting reads were then mapped to the mm10 mouse reference genome without alternate contigs using minimap2 v.2.11 with default settings for alignment of nanopore reads (-x map-ont). Reads that mapped with a quality score <50 were then filtered out using samtools v.1.7. CpG and GpC methylation were simultaneously called on the remaining reads using nanopolish v.0.11.1. Average CpG methylation was calculated for each group of CpGs that did not contain any GCGs. To visualize the methylation patterns of the reads with IGV v.2.12.2, we modified the individual reads in the alignment (BAM) files. All cytosines called as unmethylated were converted to thymine to simulate bisulfite conversion. This was achieved using code adapted from the Timp Laboratory’s nanopore-methylation-utilities (https://github.com/ timplab/nanopore-methylation-utilities). Once these converted files were loaded in IGV, we were then able to visualize methylation using IGV’s bisulfite mode. Tiled data file tracks showing aggregated CpG methylation levels were generated with igvtools v.2.4.16.
Assembly: mm10
Supplementary files format and content: methylation calls(tsv)
Library strategy: nanopore-methylation
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| Submission date |
Aug 07, 2023 |
| Last update date |
Aug 07, 2023 |
| Contact name |
Qiangzong Yin |
| E-mail(s) |
[email protected]
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| Organization name |
UMass Chan Medical School
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| Department |
Biochemistry and Molecular Biotechnology
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| Lab |
Dr. Oliver Rando's lab
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| Street address |
364 Plantation St LRB940
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL24973 |
| Series (2) |
| GSE226689 |
Revisiting chromatin packaging in mouse sperm |
| GSE240249 |
Revisiting chromatin packaging in mouse sperm [Nanopore-methylation] |
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| Relations |
| BioSample |
SAMN36876518 |
| SRA |
SRX21284026 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7688875_Sperm_untreated_NanoNOME_Whole_genome_methylation_calls.tsv.gz |
3.5 Gb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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