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| Status |
Public on Jun 11, 2024 |
| Title |
P4-mDRIP-seq-htl1Δ |
| Sample type |
SRA |
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| Source name |
htl1<delta>
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: htl1<delta>
genotype: MATa his3<delta>1 leu2<delta>0 met15<delta>0 ura3<delta>121 htl1<delta>
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For mammal and E. coli genomic DNA extraction, cells were lysed by 0.5% SDS and digested with 0.1mg/mL proteinase K (TransGen Biotech#: GE201-01) at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method. For Arabidopsis and rice gDNA extraction, seedlings were grounded in liquid nitrogen and then lysed by 0.5% SDS and digested with 0.1 mg/mL proteinase K (TransGen Biotech#: GE201-01) at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method. For yeast gDNA extraction, cells were resuspended in TE buffer with 0.5% NP40 and equal volume glass beads (Sigma#: G8772), and beat 6 times in 30 sec at 60 Hz with 1 min pausing at 4°C to destroy cell walls. Then cells were collected, lysed by 0.5% SDS and digested with 0.1mg/mL proteinase K at 37°C in a shake with 220 rpm for 4-6 hours followed by phenol-chloroform nucleic acid extraction method.
For cDNA hybrids synthesis, total RNA was extracted according to the manual of TRIzol™ (ThermoFisher Scientific#:15596026) from the manufacturer. DNase I (ThermoFisher Scientific#: EN0521) treatment was performed at 37°C to remove potential gDNA contamination. cDNA hybrids were synthesized by ProtoScript® II Reverse Transcriptase with d(T)23VN primer according to the manufacturer’s manual. Then, cDNA hybrids were incubated with MBN at 30°C for 30 min to remove remaining primers and RNA while only production with DNA:RNA hybrid form can be retained.
Nucleic acid was digested with MBN at 30°C for 1 hour and purified using the phenol-chloroform nucleic acid extraction method followed by resuspending the pellet in 130 μl TE buffer. Sonication with Covaris™ ME220 (Covaris, 70 W, 20% Duty factor, 1000 cycles per burst, 130 s, at 4–20°C) was performed to fragment nucleic acid with peak size around 250 bp and quantified by Qubit™ dsDNA HS Assay Kit (ThermoFisher Scientific#:Q32851). For multiple samples that would be used as a pool for DRIP, sonicated DNA was diluted to the needed concentration and barcoded in DNA:RNA hybrids tailing and ligation reaction for each sample as above mentioned at 37°C for 1 hour with barcode adaptors (Table S2) followed by adding 2 μL 0.5 M EDTA to stop the reaction. Then, these barcoded samples were pooled together and purified using the phenol-chloroform nucleic acid extraction method. The purified nucleic acid pellet was resuspended in TE buffer followed by DRIP and library construction for sequencing as described in ssDRIP-seq.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
Raw reads with low quality and adaptor contamination were trimmed, and short reads less than 50 pb were filtered by Trim Galore (version 0.6.7).
After trimming, clean reads were aligned to the genomes (BW25113 genome for E. coli, sacCer3 genome for yeast, GRCh38 genome for humans, mm10 genome for mice, TAIR10 genome for Arabidopsis, MSU version 7.0 for rice) with Bowtie 2 (version 2.2.5) using local alignment mode.
Assembly: sacCer3 genome for yeast
Supplementary files format and content: bigWig
Library strategy: mDRIP-seq
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| Submission date |
Jun 21, 2023 |
| Last update date |
Jun 11, 2024 |
| Contact name |
Changbin Sun |
| Organization name |
Chinese Academy of Agricultural Sciences
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| Department |
Agricultural Genomics Institute at Shenzhen
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| Lab |
Guangdong Laboratory of Lingnan Modern Agriculture
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| Street address |
Dapeng
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| City |
Shenzhen |
| ZIP/Postal code |
518120 |
| Country |
China |
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| Platform ID |
GPL27812 |
| Series (2) |
| GSE219068 |
mDRIP-seq: a high-throughput method for R-loop mapping and quantitative assessment |
| GSE219071 |
mDRIP-seq is a high-throughput method for quantitative R-loop landscape profiling |
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| Relations |
| BioSample |
SAMN35823127 |
| SRA |
SRX20742579 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7503376_P4-htl1_RPGC_cRloop.bw |
1.8 Mb |
(ftp)(http) |
BW |
| GSM7503376_P4-htl1_RPGC_wRloop.bw |
2.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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