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| Status |
Public on Jan 31, 2024 |
| Title |
CseDa01_dsDNA_rep4 |
| Sample type |
SRA |
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| Source name |
E. coli C2566, phage lambda, plasmid pRSSM1.PleII, phage XP12, phage T4 AGT-
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| Organism |
Escherichia coli |
| Characteristics |
cell type: E. coli C2566, phage lambda, plasmid pRSSM1.PleII, phage XP12, phage T4 AGT-
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
E. coli C2566 genomic DNA was purified using Monarch Genomic DNA Purification Kit (NEB Ipswich, MA), GM12878 genomic DNA was obtained from Coriell Cell Repositories (Camden, NJ).
Input DNA was sheared to 300 bp using the Covaris S2 instrument. Sheared material was transferred to a PCR strip tube to begin library construction. NEBNext DNA Ultra II Reagents (NEB, Ipswich, MA) were used according to the manufacturer’s instructions for end repair, A-tailing, and adaptor ligation. The custom made Pyrollo-dC adaptor (NEB Organic Synthesis Division, Ipswich MA), where all dCs are replaced with Pyrollo-dC, was used. The ligated samples were purified using NEBNext Sample Purification Beads according to the manufacturer’s instructions. For ssDNA libraries, prior to deamination the DNA was denatured by heating at 90oC for 10 minutes followed by cooling for 2 minutes on ice. The DNA was then deaminated using 1 µL of PURExpress (NEB, Ipswich, MA) synthesized deaminase protein following manufacturer's recommendations for 1 hour at 37oC. Then 1 µL of Thermolabile Proteinase K (NEB, Ipswich, MA) was added and incubated for 30 min at 37oC followed by 10 min at 60oC. Then the library was amplified using NEBNext Q5U Master Mix (NEB, Ipswich, MA, USA). Paired-end sequencing of 150 cycles (2 x 75 bp) was performed for all the sequencing runs.
DNA sequencing of deaminated double-stranded and single-stranded DNA
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
basecalls were performed using Illumina RTA software and fastq files were generated from the BCL files using Picard’s basecallstofastq.
Reads were trimmed for adaptor sequence and low quality bases at the 3' end using the trim_galore program.
The trimmed read sequences were C to T converted and were then mapped to the a composite reference including E.coli and all the control sequences using the Bismark program with the default Bowtie2 settings.
The first 5 bp at the 5’ end of R2 reads were removed to reduce end-repair errors and aligned read pairs that shared the same alignment ends were regarded as PCR duplicates and were discarded.
The numbers of Ts (converted not methylated) and Cs (unconverted modified) of each covered cytosine position were then calculated from the remaining good quality alignments using Bismark methylation extractor.
Assembly: E. coli C2566, human hg38
Supplementary files format and content: tab-delimited text/bed files include genomic coordinates, number of Cs and number of Ts and sequence contexts of covered cytosines.
Library strategy: SEM-seq
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| Submission date |
Jan 09, 2024 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Zhiyi Sun |
| E-mail(s) |
sunz@neb.com
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| Organization name |
New England Biolabs
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| Department |
Research
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| Street address |
240 County Road
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| City |
Ipswich |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01938 |
| Country |
USA |
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| Platform ID |
GPL32081 |
| Series (1) |
| GSE233932 |
Discovery of diverse DNA cytosine deaminases enables a single-enzyme method for base resolution methylation detection |
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| Relations |
| BioSample |
SAMN39325619 |
| SRA |
SRX23146887 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8009290_CseDa01_dsDNA_rep4_4mC_plasmid.info.txt.gz |
36.1 Kb |
(ftp)(http) |
TXT |
| GSM8009290_CseDa01_dsDNA_rep4_C_ecoli.info.txt.gz |
18.9 Mb |
(ftp)(http) |
TXT |
| GSM8009290_CseDa01_dsDNA_rep4_ghmC_T4AGT-.info.txt.gz |
499.6 Kb |
(ftp)(http) |
TXT |
| GSM8009290_CseDa01_dsDNA_rep4_mC_XP12.info.txt.gz |
383.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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