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| Status |
Public on May 29, 2024 |
| Title |
Zebrafish, Sperm2 |
| Sample type |
SRA |
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| Source name |
sperm
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| Organism |
Danio rerio |
| Characteristics |
genotype: Wild type
ecotype: Tubingen
developmental stage: sperm
pulldown of: DNA:RNA hybrid
protocol: ULI-ssDRIP-seq with spike-in
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples collected were lysed in genome extraction buffer (200 mM NaCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS, 200 μg /ml freshly added Proteinase K) at 37°C for 8~12 h. Then, 1/4 volume of 5 M potassium acetate solution (pH 5.2) was added to the lysate and mixed gently, followed by incubation on ice for 20 min. Following extraction with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, pH 7.8) and chloroform sequentially with phase-lock tubes. The supernatant containing genomic DNA was precipitated by addition of equal volume of isopropanol and 1 μl GlycoBlue (Thermo Fisher) for 30 min at -20°C. The precipitate was washed by 70% ethanol for once and then air-dried.
Genomic DNA was digested with mung bean nuclease (TaKaRa) at 37°C for 2 h and purified by the phenol-chloroform method followed by resuspending the pellet in 130 μl TE buffer. Digested gDNA was sonicated to a peak fragment size of 250 bp, performed on a S220 Focused-ultrasonicator with 10% Duty Factor, 200 cycles/burst, 175 peak incident power and for 90 sec per tube. For inter-group relative quantification, gDNA from Arabidopsis (7-day-old Col-0 seedlings) was used as the spike-in sample to normalize total R-loop levels between animal samples. Sonicated spike-in gDNA to a peak fragment size of 250 bp as described above, and then added ~0.1 ng sonicated spike-in gDNA to sonicated target gDNA samples. 80% of the mixed sample was used for the first adapter ligation of ULI-ssDRIP-seq workflow, and the rest 20% part was used for input-library preparation by using Accel-NGS 1S Plus DNA Library Kit following the manual. For the first adapter ligation (pre-ligation), 53 μl sonicated gDNA was added to Adaptase reaction mix (for each sample: 8 μl buffer G1, 8 μl reagent G2, 5 μl reagent G3, 2 μl enzyme G4, 2 μl enzyme G5, and 2 μl enzyme G6, from Accel-NGS 1S Plus DNA Library Kit), incubated at 37°C for 1 hour. Then DRIP was performed as described previously 5. Prepared 174 ul pre-mixed extension reaction mix following the extension part of manual (Accel-NGS 1S Plus DNA Library Kit) and mixed with beads/antibody complexes, followed by 1 cycle PCR (98°C 90 sec, 63°C 30 sec, 68°C 5 min). Purified DNA by adding 1.2 volumes of SPRIselect beads. Ligated the second truncated adapter to the 5’ ends at 25°C for 1 hour following the ligation part of manual (Accel-NGS 1S Plus DNA Library Kit). Purified DNA again by 1 volume SPRIselect beads and eluted with 21 ul Low EDTA buffer. 1 ul purified DNA was used for qPCR by using indexing primers to address cycle numbers of indexing PCR. The cycle numbers of indexing PCR was equal to the Ct value. An indexing PCR step was performed following the manual of Accel-NGS® 1S Plus DNA Library Kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: DRIP-seq
For basic or modified ssDRIP-seq, reads were aligned to the Arabidopsis TAIR10 genome with Bowtie 2 (version 2.3.4.2) using default settings. For ULI-ssDRIP-seq without spike-in, reads were aligned to the zebrafish danRer7 genome with Bowtie 2. Duplicates were removed by Picard tools (http://broadinstitute.github.io/picard). Using samtools (version 1.3), unmapped reads and reads with more than three mismatches were removed, and the total mapped reads (non-strand R-loops) were divided into wR-loop and cR-loop reads (wR-loop means an R-loop with a Watson strand unpaired ssDNA, while cR-loop means an R-loop with a Crick strand unpaired ssDNA) . Aligned reads files (BAM) were converted to normalized coverage files (bigWig) by using deepTools (version 2.4.2). Read coverage of data from basic or modified ssDRIP-seq, or ULI-ssDRIP-seq without spike-in, was normalized to 1× sequencing depth (also known as Reads Per Genomic Content, RPGC). For quantitative ULI-ssDRIP-seq, alignment and normalization steps were different from those described above. Reads from ULI-ssDRIP-seq or input library were aligned to the zebrafish danRer7 or Arabidopsis TAIR10 genome with Bowtie 2 respectively. Normalization: First, calculated read count of ULI-ssDRIP-seq or input library mapped to danRer7 (target sample [T]) or TAIR10. (spike-in sample [S]). Tn or T(n) represented target n, while Sn or S(n) represented spike-in n (n = 1, 2, 3…, represent each sample). Choose any sample as inter-sample normalization control (represented by T1; in this study, muscle rep1 was chosen as T1), and calculated its normalization factor of reads mapped to danRer7 in RPGC manner described above. T(n)R-loop represented read count of ULI-ssDRIP-seq library from T(n) mapped to danRer7, while S(n)input represented read count of input library from S(n) mapped to TAIR10. By analogy, T(n)input represented read count of input library from T(n) mapped to danRer7, while S(n)R-loop represented read count of ULI-ssDRIP-seq library from S(n) mapped to TAIR10. The quantitative factor of Tn equaled [T(n)R-loop × S(n)input] / [ S(n)R-loop × T(n)input]. The finial normalization factor of Tn equaled Quantitative factor (Tn) multiplied by Normalization factor (T1) divided by Quantitative factor (T1). For other samples except T1, the finial normalization factor was used for normalization to convert bigWig file.
Genome_build: TAIR10
Supplementary_files_format_and_content: bigwig
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| Submission date |
Sep 05, 2021 |
| Last update date |
May 29, 2024 |
| Contact name |
li kuan |
| E-mail(s) |
likuan@cibr.ac.cn
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| Organization name |
Tsinghua University
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| Street address |
zhongguancun
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| City |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE183453 |
R-loop landscapes during parental-to-zygotic transition in zebrafish |
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| Relations |
| BioSample |
SAMN21237711 |
| SRA |
SRX12019712 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5557861_Sperm2_cRloop.bw |
102.8 Mb |
(ftp)(http) |
BW |
| GSM5557861_Sperm2_wRloop.bw |
102.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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