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| Status |
Public on Apr 24, 2023 |
| Title |
control pUC19, lambda phage, and T4-phage, no_TET_no_deam |
| Sample type |
SRA |
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| Source name |
control pUC19, lambda phage, and T4-phage
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| Organisms |
Cloning vector pUC19; Escherichia phage T4; Escherichia phage Lambda |
| Characteristics |
sample type: control pUC19, lambda phage, and T4-phage
treatment: no TET
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro derived controls for all other samples.
1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, fully 5mCpG modified Lambda control gDNA, and fully 5hmC-modified T4 phage gDNA) was used for each condition. DNA was added to a 20 μL reaction containing 1x NEB CutSmart buffer (50 mM Potassium Acetate 20 mM Tris-acetate, 10 mM Magnesium Acetate, and 100 μg/ml BSA), 0.04 nM UDP-glucose, and 10 U T4-βGT. The glucosylated DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The purified DNA was then in a 50 μL reaction containing 1x NEB EM-Seq TET buffer (50 mM Tris pH 8.0, 1 mM DTT, 5 mM sodium-L-ascorbate, 20 mM αKG, 2 mM ATP, and 50 mM ammonium iron (II) sulfate hexahydrate) and 16 μg TET2 (NEB). The reaction was then incubated at 37°C for 80 min. Following oxidation, 0.8 U of Proteinase K (NEB) was added and the mixture was incubated for 30 min at 37°C. The oxidized DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The no TET control was carried through mock oxidation reactions without TET enzyme. The DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The purified oxidized sample was then end-prepped and ligated with the same protocol as above except for the adapters used, which was different for each method (no deamination, TAPS, TAPS-β: C adapters, BS-Seq: 5mC adapters, DM-Seq: 5pyC adapters). BS deamination, A3A deamination, pyridine borane deamination, or a no deamination control were then performed.
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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: pUC19, lambda phage ViralProj14204, T4 phage ViralProj14044
Supplementary files format and content: For control DNA samples where read level information for each control genome was analyzed, aligned files (.bam) are provided, not deduplicated and not filtered. The .bam format makes the number of reads mapping to each genome as well as data reporting percent modification accessible.
Library strategy: no deamination
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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 |
GPL33181 |
| Series (2) |
| GSE225973 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [4] |
| GSE225975 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution |
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| Relations |
| BioSample |
SAMN33426277 |
| SRA |
SRX19487485 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7061150_no_TET_no_deam.bam |
2.4 Mb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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