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| Status |
Public on Apr 24, 2023 |
| Title |
control lambda phage, unmeth_DM |
| Sample type |
SRA |
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| Source name |
control lambda phage
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| Organism |
Escherichia phage Lambda |
| Characteristics |
sample type: control lambda phage
treatment: unmethylated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro derived controls for all other samples.
Lambda gDNA was either unmethylated, CpG methylated, or GpC methylated.
10 ng of sheared lambda gDNA ligated to 5pyC-containing adapters was used as input for DM-Seq when only comparing to BS-Seq. A methylated copy strand was created. 1 μM fully-methylated copy primer was annealed in a total volume of 10 μL in CutSmart Buffer and 1 mM final concentration (individually) of dATP/dGTP/dTTP (Promega) and dmCTP (NEB). 1 μl or 8 units Bst polymerase, large fragment (NEB) was added and incubated for 30 min at 65°C. The 5hmCs were then glucosylated with 40 μM UDP-Glucose and 1 μL or 10 units of T4 Phage β-glucosyltransferase (NEB) for 1 hour at 37°C in a final volume of 20 μL. Incompletely copied or uncopied fragments were degraded with 1 μL or 10 units Mung Bean Nuclease (NEB) for 30 min at 30°C. After SPRI magnetic bead purification (1.2x), libraries were mixed with 0.5 μM MBP-M.MpeI-N374K and 160 μM CxSAM in carboxymethylation buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated overnight at 37°C followed by denaturation for 5 min at 95°C. 1 μL or 0.8 units of Proteinase K (NEB) was subsequently added and incubated at 37°C for 15 min. The samples were purified using SPRI magnetic beads (1.2x) and eluted in 1 mM Tris-Cl, pH 8.0. DNA was then subjected to snap-cooling and A3A deamination in a final volume of 50 μL as previously described before SPRI magnetic beads purification (1.2x). DM-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X). Libraries were then characterized using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).
BS-Seq was performed on 10 ng of sheared lambda gDNA ligated to 5mC-containing adapters (xGen, IDT), when only comparing to DM-Seq, with no added copy or DM-Seq specific steps, using manufacturer instructions (Diagenode). Purified BS-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X) and ultimate characterization using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
For all workflows, reads were quality and length trimmed with Trim Galore! Reads were aligned with Bismark and deduplicated with Picard. All data was analyzed single-end. DM-Seq reads were filtered if 3 consecutive CpHs were non-converted using Bismark’s existing filter_non_conversion command. Only reads with MAPQ ≥ 30 were analyzed.
Assembly: lambda phage ViralProj14204
Supplementary files format and content: Tab-delimited text files (.txt) include genomic coordinates (chr, start, and end) and deamination (BS-Seq, DM-Seq) analysis (signal [C/(C+T)], sequencing coverage, and strand) of all annotated CpG sites in control genomes. They additionally have the +1 and +2 base identity.
Library strategy: DM-Seq
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| Submission date |
Feb 23, 2023 |
| Last update date |
Apr 25, 2023 |
| Contact name |
Rahul M. Kohli |
| E-mail(s) |
kohlilab@gmail.com
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| Phone |
215-573-7523
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| Organization name |
University of Pennsylvania
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| Department |
Department of Medicine
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| Lab |
Rahul M. Kohli
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| Street address |
3610 Hamilton Walk, 502B Johnson Pavilion
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL33180 |
| Series (2) |
| GSE225967 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [2] |
| GSE225975 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution |
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| Relations |
| BioSample |
SAMN33426361 |
| SRA |
SRX19487553 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7061104_unmeth_DM.txt.gz |
33.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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