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Sample GSM7061103 Query DataSets for GSM7061103
Status Public on Apr 24, 2023
Title control lambda phage, unmeth_BS
Sample type SRA
 
Source name control lambda phage
Organism Escherichia phage Lambda
Characteristics sample type: control lambda phage
treatment: unmethylated
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).
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina MiSeq
 
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.
 
Submission date Feb 23, 2023
Last update date Apr 25, 2023
Contact name Rahul M. Kohli
E-mail(s) kohlilab@gmail.com
Phone 215-573-7523
Organization name University of Pennsylvania
Department Department of Medicine
Lab Rahul M. Kohli
Street address 3610 Hamilton Walk, 502B Johnson Pavilion
City Philadelphia
State/province Pennsylvania
ZIP/Postal code 19104
Country USA
 
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
Relations
BioSample SAMN33426362
SRA SRX19487552

Supplementary file Size Download File type/resource
GSM7061103_unmeth_BS.txt.gz 28.0 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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