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| Status |
Public on Mar 15, 2024 |
| Title |
embryo, 62h, rep1 |
| Sample type |
SRA |
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| Source name |
Embryo
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| Organism |
Ascaris suum |
| Characteristics |
tissue: Embryo
developmental stage: 62h
treatment: FseI
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ascaris females were collected, and the fertilized embryos (0hr, 1-cell before prenuclear fusion) were harvested and processed as previously described (Wang et al. 2011, Wang et al. 2014). Ascaris 0hr samples were incubated at 30°C with constant shaking for the desired time (from 50hr to 98hr). For all molecular experiments, the eggshells were first de-coated with bleach treatment (0.4 M KOH, 2% sodium hypochlorite) for 1.5 hours at 30°C. Parascaris samples were collected as described (Simmons et al. 2023, Wang et al. 2017). Parascaris eggs were prepared similarly to Ascaris, except the incubation was carried out at 37°C and the embryonation time was shorter (10-14hr).
An adapted END-seq protocol was used (Canela et al. 2016, Wong et al. 2021). Embryos were embedded in agarose plugs. Some plugs were treated with the restriction enzymes as internal controls. Plugs were treated with exonuclease VII and exonuclease T. Blunt ends were A-tailed and capped with END-seq adaptor 1 (Canela et al. 2016). Plugs were melted and DNA was sheared to 200-300 bp with a Covaris M220 focused ultrasonicator. DNA fragments containing END-seq adaptor 1 were isolated with Dynabeads MyOne Streptavidin C1. END-seq adaptor 2 was ligated to the sheared ends of the A-tailed DNA fragments. The hairpins within the adaptors were digested with USER, and the DNA was amplified with Illumina TruSeq primers and barcodes. The following modifications were made to the END-seq protocol to capture break ends with different features. For the direct capture method, we excluded the exonuclease VII and exonuclease T treatments. For the all-END protocol, the plugs were treated with T4 polymerase (with dNTPs).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Only read 1 (R1) files were processed. Reads containing two sequential telomeric repeats (TTAGGCTTAGGC or GCCTAAGCCTAA) were filtered from the data using an in-house Perl script
Reads were mapped to the appropriate genome with Bowtie2 (local alignment) and processed with SAMtools
For samples treated with restriction enzyme BEDTools intersect was used to remove reads mapped to restriction sites
The 5’-end position of each read was mapped, separated by strand, and normalized to ten million genome-mapped reads using BEDtools genomecov
The mapping results were converted to bigWig format using bedGraphToBigWig
To analyze new telomere addition during PDE developmental stages, END-seq reads containing two consecutive telomere repeats (filtered in step 1) were converted to the G-rich strand (TTAGGC).
Reads were mapped to the genome with Bowtie2 end-to-end alignment
Mapped reads were false-positive telomeric reads. Unmapped reads were trimmed with fastx_clipper “-v -n -l 25 -a TTAGGCTTAGGC” parameters.
Trimmed reads were mapped to the genome with bowtie2 (local alignment) and processed as described above except except the 3’-positions (the sites where new telomeres are added) rather than 5’-positions were obtained with BEDTools genomecov
Assembly: Ascaris suum ASM1343314v1, Parascaris univalens (Pu_v2)
Supplementary files format and content: bigWig file containing the 5' end of reads on the forward strand
Supplementary files format and content: bigWig file containing the 5' end of reads on the reverse strand
Supplementary files format and content: bigWig file with the 3' end of reads on the forward strand which contained two telomeric repeats (TTAGGCTTAGGC)
Supplementary files format and content: bigWig file with the 3' end of reads on the reverse strand which contained two telomeric repeats (TTAGGCTTAGGC)
Library strategy: END-seq
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| Submission date |
Mar 06, 2024 |
| Last update date |
Mar 15, 2024 |
| Contact name |
Jianbin Wang |
| E-mail(s) |
jianbin.wang@utk.edu
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| Phone |
865-974-4085
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| Organization name |
University of Tennessee Knoxville
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| Department |
Biochemistry & Cellular and Molecular Biology
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| Lab |
420C
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| Street address |
1311 Cumberland Avenue
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| City |
Knoxville |
| State/province |
TN |
| ZIP/Postal code |
37996 |
| Country |
USA |
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| Platform ID |
GPL31097 |
| Series (1) |
| GSE260958 |
End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination |
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| Relations |
| BioSample |
SAMN40278994 |
| SRA |
SRX23849719 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8128716_endSeq_62h_Rep1_FseI.ends.fwd.bw |
10.3 Mb |
(ftp)(http) |
BW |
| GSM8128716_endSeq_62h_Rep1_FseI.ends.rev.bw |
10.2 Mb |
(ftp)(http) |
BW |
| GSM8128716_endSeq_62h_Rep1_FseI.telomere.ends.fwd.bw |
227.3 Kb |
(ftp)(http) |
BW |
| GSM8128716_endSeq_62h_Rep1_FseI.telomere.ends.rev.bw |
239.9 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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