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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 01, 2024 |
| Title |
mESC E14, nuclei, m6dA methylated, ex situ SAMOSA-Tag |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cell E14
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| Organism |
Mus musculus |
| Characteristics |
cell line: mouse embryonic stem cell E14
cell type: mouse embryonic stem cell
treatment: m6dA methylated
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| Treatment protocol |
Isolated mESC E14 nuclei were methylated (10uL EcoGII per 1-2e6 nuclei) with non-specific adenine methyltransferase EcoGII (New England Biolabs, HC stock 2.5e4U/mL) at 37°C for 30 minutes, supplemented with SAM to 1.16mM (NEB) after 15 minutes.
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| Growth protocol |
Feeder-free E14 mouse embryonic stem cells (mESC E14) cultures were maintained on 0.2% gelatin, in KnockOut DMEM 1X (Gibco) supplemented with 10% Fetal Bovine Serum (Phoenix Scientific), 1% 100X GlutaMAX (Gibco), 1% 100X MEM Non-Essential Amino Acids (Gibco), 0.128mM 2-mercaptoethanol (BioRad), and 1X Leukemia Inhibitory Factor. Cultures were passaged at least twice before use.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tagmented nuclei were pre-treated with 10uL RNaseA at 37°C for 15 minutes, then purified with 2.65uL Proteinase K (20mg/mL), and 2.65uL 10% SDS at 65°C for 3 hours. DNA was extracted using an equal volume of phenol:chloroform:isoamyl Alcohol (25:24:1, v/v), followed by 100% ethanol percipitation overnight at -80ºC.
Extracted DNA was tagmented using SMRT-Tag conditions (in 1X Tagmentation Mix (10mM TAPS-NaOH pH 8.5, 5mM MgCl2, and 10% DMF) , 55ºC for 30 min) with 0.046 pmol H-Tn5-R27S,E54K,L372P (QB3 MarcoLab) , then terminated by adding 0.2% SDS (final concentration 0.04%). Tagmented fragments were gap-repaired using Phusion DNA polymerase and Taq Ligase at 37ºC for 1hr, digested with Exonuclease III at 37ºC for 1hr, then cleaned up using Ampure PB. Library quality was assessed with Qubit 1x High Sensitivity DNA Assay and Agilent Bioanalyzer High Sensitivity DNA kit.
OTHER: Ex situ SAMOSA-Tag Library Preparation
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel II |
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| Data processing |
Demultiplexing subreads BAM files with lima (v2.6.0)
Generating circular consensus sequences (CCS) using ccs (v6.4.0) with kinetic information
Extracting kinetic information from CCS reads using the SAMOSA-ChAAT computational pipeline
Aligning CCS reads to the mm10 reference genome using pbmm2 (v.1.9.0)
Assembly: mm10
Supplementary files format and content: *_full.pickle: Python pickle files containing IPD measurements for each base in the forward and reverse strands per CCS read
Supplementary files format and content: *_zmwinfo.pickle: Python pickle containing a dataframe with length, # of passes, and mean IPD per base per CCS read
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| Submission date |
Oct 20, 2023 |
| Last update date |
May 01, 2024 |
| Contact name |
Arjun S Nanda |
| Organization name |
Gladstone Institutes / UCSF
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| Department |
Gladstone Institute for Data Science & Biotechnology
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| Lab |
Ramani Lab
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| Street address |
1700 Owens St
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| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL29177 |
| Series (2) |
| GSE225314 |
Direct transposition of native DNA for sensitive multimodal single-molecule sequencing |
| GSE245946 |
Direct transposition of native DNA for sensitive multimodal single-molecule sequencing IV |
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| Relations |
| BioSample |
SAMN37908750 |
| SRA |
SRX22158394 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7851673_SAMOSA-Tag.subread.E14_ex_situ_100ng_full.pickle.gz |
1.7 Gb |
(ftp)(http) |
PICKLE |
| GSM7851673_SAMOSA-Tag.subread.E14_ex_situ_100ng_full_zmwinfo.pickle.gz |
1.7 Mb |
(ftp)(http) |
PICKLE |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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