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| Status |
Public on Aug 07, 2020 |
| Title |
GM12878 nanoNOMe |
| Sample type |
SRA |
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| Source name |
nanoNOMe on GM12878 Cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GM12878
cell type: EBV-transformed lymphoblastoid cell line
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| Treatment protocol |
NOMe-seq was performed on the cells with adjustments for nanopore sequencing. Cells were collected by trypsinization, then nuclei were extracted by incubating in resuspension buffer (100 mM Tris-Cl, pH 7.4, 100 mM NaCl, 30 mM MgCl2) with 0.25 % NP-40 for 5 minutes on ice. Intact nuclei were collected by centrifugation for 5 minutes at 500xg at 4 °C. Nuclei were subjected to a methylation labeling reaction using a solution of 1x M. CviPI Reaction Buffer (NEB), 300 mM sucrose, 96 μM S-adenosylmethionine (SAM; New England Biolabs, NEB), and 200 U M. CviPI (NEB) in 500 μL volume per 500,000 nuclei. The reaction mixture was incubated at 37 °C with shaking on a thermomixer at 1,000 rpm for 15 minutes. SAM was replenished at 96 μM at 7.5 minutes into the reaction. The reaction was stopped by the addition of an equal volume of stop solution (20 mM Tris-Cl, pH 7.9, 600 mM NaCl, 1% SDS, 10 mM disodium EDTA).
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| Growth protocol |
Cell lines were grown according to manufacturer's specifications
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were treated with proteinase K (NEB) at 55 °C for > 2 hours, and DNA was extracted via phenol:chloroform extraction and ethanol precipitation. After proteinase K treatment, and in all following steps, samples were handled with care using large orifice pipette tips to avoid excessive fragmentation of DNA.
Purified gDNA was prepared for nanopore sequencing following the protocol in the genomic sequencing by ligation kit LSK-SQK108 (ONT). Samples were first sheared to ~10 kb using G-tubes (Covaris): by centrifuging 2-3 μg of unfragmented gDNA at 5,000x g for 1 minute, then inverting the tube and centrifuging again. We sheared the DNA to 10 kb because it produces long fragments of DNA while maximizing the yield of nanopore sequencing. Shearing to larger sizes or unsheared DNA may be used to maximize the length of sequenced reads, with the caveat that sequencing yield will drop. In two samples (GM12878 samples 8 and 9), we targeted 20kb fragments, with an additional step of removing short fragments using the Short Read Eliminator module by Circulomics, following the manufacturer’s specifications. The sheared samples were end-repaired and dA-tailed using NEBnext Ultra II end-repair module (NEB), followed by clean-up using 1x v/v AMPure XP beads (Beckman Coulter). Sequencing adaptors, comprised of leader adaptor DNA and motor proteins, were ligated to the end-prepared DNA fragments using Blunt/TA Ligase Master Mix (NEB), followed by clean-up using 0.4x v/v AMPure XP beads and sequencing kit reagents. >400 ng of adaptor ligated samples per flow cell were loaded onto FLO-MIN106 or PRO-002 flowcells and run on MinION Mk1b, GridION, or PromethION sequencers for up to 72 hours.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
PromethION |
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| Data processing |
Library strategy: Oxford Nanopore Sequencing (NOMe-seq)
Raw current signals were converted to DNA sequences using Guppy version 3.0.3 (ONT), using the “high-accuracy” basecalling model.
DNA sequences were aligned to hg38 human reference genome without alternative contigs using NGMLR version 0.2.8 with default settings for aligning Oxford nanopore reads (-x ont)
CpG and GpC methylation were called using nanopolish version 0.11.1.
Nanopolish outputs were converted to methylation bed files (one line per read) as specified by https://github.com/isaclee/nanopore-methylation-utilities using mtsv2bedGraph.py
Methylation bed files were subsequently converted to bismark-style methylation counts (one line per locus) using parseMethylbed.py frequency in https://github.com/isaclee/nanopore-methylation-utilities
Single-read anallysis was performed on select genomic contexts (CTCF binding sites and gene promoter regions), and the resulting data was deposited into Zenodo (https://zenodo.org/record/3969567)
Genome_build: hg38
Supplementary_files_format_and_content: Methylation Counts files contain the counts of methylation states for each observed genomic locus in the same format as bismark cytosine reports : <chromosome> <position> <strand> <count methylated> <count unmethylated> <C-context> <trinucleotide context> (https://rawgit.com/FelixKrueger/Bismark/master/Docs/Bismark_User_Guide.html)
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| Submission date |
Aug 06, 2020 |
| Last update date |
Aug 07, 2020 |
| Contact name |
Isac Lee |
| E-mail(s) |
[email protected]
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| Organization name |
Johns Hopkins University
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| Street address |
3400 N. Charles St.
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21218 |
| Country |
USA |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE155791 |
Methylation and accessibility profiling of GM12878, MCF-10A, MCF-7, and MDA-MB-231 using nanopore sequencing |
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| Relations |
| BioSample |
SAMN10614997 |
| SRA |
SRX8465925 |
| SRA |
SRX8465924 |
| SRA |
SRX5354365 |
| SRA |
SRX5354364 |
| SRA |
SRX5354363 |
| SRA |
SRX5354362 |
| SRA |
SRX5354361 |
| SRA |
SRX5354360 |
| SRA |
SRX5354359 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4712207_GM12878_nanoNOMe.cpg.mfreq.txt.gz |
134.5 Mb |
(ftp)(http) |
TXT |
| GSM4712207_GM12878_nanoNOMe.gpc.mfreq.txt.gz |
632.6 Mb |
(ftp)(http) |
TXT |
| GSM4712207_GM12878_nanoNOMe.hap1.cpg.mfreq.txt.gz |
119.2 Mb |
(ftp)(http) |
TXT |
| GSM4712207_GM12878_nanoNOMe.hap1.gpc.mfreq.txt.gz |
561.5 Mb |
(ftp)(http) |
TXT |
| GSM4712207_GM12878_nanoNOMe.hap2.cpg.mfreq.txt.gz |
119.2 Mb |
(ftp)(http) |
TXT |
| GSM4712207_GM12878_nanoNOMe.hap2.gpc.mfreq.txt.gz |
561.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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