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Status |
Public on Aug 09, 2024 |
Title |
P28 cortical neurons Nanopore Sequencing rep2 |
Sample type |
SRA |
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Source name |
cortex
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Organism |
Mus musculus |
Characteristics |
tissue: cortex cell type: neuron genotype: WT developmental stage: P28
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Extracted molecule |
genomic DNA |
Extraction protocol |
High molecular weight (HMW) DNA was extracted from isolated cells using the Monarch HMW DNA extraction kit (New England Biolabs, #T3050L) following the manufacturer’s instructions. The HMW DNA was sheared to 20kb with g-TUBE (Covaris #520079). Library preparation was following Nanopore protocols to attach sequencing adapters to the DNA ends with Ligation Sequencing Kit (Oxford Nanopore Technologies, #SQK-LSK110). One flow cell was applied for each library to ensure an optimal sequencing depth of around 20x coverage at each cytosine and sequenced on PromethION (Steger Hall sequencing core, Virginia Tech).
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
PromethION |
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Description |
Methylation_5mC_sites_cov9.bed.gz Methylation_5hmC_sites_cov9.bed.gz DMS_Astrocyte_Neuron_5mC_cov9.bed.gz DMS_Neuron_Microglia_5mC_cov9.bed.gz DMS_Astrocyte_Neuron_5hmC_cov9.bed.gz DMS_Neuron_Microglia_5hmC_cov9.bed.gz
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Data processing |
The raw signal stored in fast5 files were converted to nucleotide sequence, which were mapped to the mouse reference genome mm10 with the tool megalodon (v 2.4.2). Quality control was performed using the tool, pycoQC (v2.5.2) with the sequencing summary file generated by Megalodon. Methylated CpG sites were distinguished based on the electronic signal difference and mapping result with the model provided by Remora (v0.1.2). The final output provides the position and quantitative methylation level of each cytosine site for both 5mc and 5hmc modification. Only CpG sites with average coverage greater than 3 and identified in at least two libraries were used for the following analysis. Methylated sites were defined as CpG sites with methylation levels over 10%. DMSs and DMRs among neurons, astrocytes and microglia were identified with the R package DSS (v2.44.0) with smoothing = T setting. Only sites with methylation level difference greater than 10% and FDR less than 0.01 were considered as DMSs. Assembly: mm10
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Submission date |
Sep 28, 2023 |
Last update date |
Aug 09, 2024 |
Contact name |
Michelle Lynne Olsen |
E-mail(s) |
molsen1@vt.edu
|
Organization name |
Virginia Tech
|
Department |
School of Neuroscience
|
Lab |
Olsen Lab
|
Street address |
970 Washington Street SW
|
City |
Blacksburg |
State/province |
VA |
ZIP/Postal code |
24060 |
Country |
USA |
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|
Platform ID |
GPL26624 |
Series (2) |
GSE244251 |
Genome-wide 5mC and 5hmC patterns determine unique transcriptional signatures,regulators and exon inclusion of neural cell types in mouse brain [Nanopore] |
GSE244256 |
Genome-wide 5mC and 5hmC patterns determine unique transcriptional signatures,regulators and exon inclusion of neural cell types in mouse brain |
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Relations |
BioSample |
SAMN37413695 |
SRA |
SRX21796929 |