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| Status |
Public on May 21, 2024 |
| Title |
RDD roX2 Rep3 saturated sequencing |
| Sample type |
SRA |
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| Source name |
S2
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| Organism |
Drosophila melanogaster |
| Characteristics |
treatment: Wild type
cell line: S2
cell type: late stage 20-24 hours old Drosophila melanogaster embryos
enriched factor: roX2
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| Treatment protocol |
For heat treatment, the exponential growth phase of cells were aliquot to three tubes, one tube was carried out crosslinking directly (Control, Ctrl), two tubes were subjected to 37 °C for 20 min and then one of them was stepped into crosslinking procedure immediately (heat shock, HS), the other was kept at room temperature for 20 min then was performed crosslinking (Recovery, RC).
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| Growth protocol |
Drosophila S2 cells were cultured in Gibco™ Schneider’s Drosophila Medium supplemented with 10% fetal bovine serum and maintained at 27 °C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Dual-crosslinked cells were subjected for cell lysis and nuclei lysis. The lysed nuclei are fragmented into smaller complexes by sonication. Then the ncRNA of interest and its associated complexes are purified by hybridization using biotinylated antisense probes of a target ncRNA. And the purified complexes are immobilized on streptavidin C1 beads via high affinity with biotin, the excess unbound sites of beads are specifically blocked with denatured biotins to avoid the adsorption of biotinylated bridge-linker by streptavidin beads in later procedure of proximity ligation. Chromatin DNA contacts of complexes on beads are captured by incorporated biotinylated linkers between DNA sites during proximity ligation. Finally the ligated products are subjected for Tn5 tagmentation and enriched by streptavidin M280 beads for sequencing to obtain the specific RNA associated chromatin DNA–DNA interactions.
The ligated DNA was fragmented by using Tn5 transposase. Then the fragmented DNA contained RDD-linker were captured with Streptavidin beads and used for PCR amplification. These PCR products were then performed double size-selection with AMPure beads to achieve the DNA fragments with size of 300-600 bp, then subjected for Illumina NovaSeq 6000 with paired-end sequencing at read lengths of 150 bp.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
RDD: We processed RDD data using modified ChIA-PIPE pipeline. It includes the following main procedures: linker filtering, data alignment and qualification, coverage generation, interaction clustering, quality control, and finally data classification for visualization and further analyzation. Briefly, a configuration file and two FASTQ files (R1 reads and R2 reads) were taken. Firstly, read pairs are scanned for the RDD-linker sequence and partitioned into categories: read pairs with (i) no linker, (ii) one linker and one usable genomic tag, or (iii) a linker and PETs. PETs are aligned to a dm3 reference genome. The analysis-ready BAM file contains uniquely mapped, nonredundant PETs. Secondly, 2D chromatin interaction maps are generated using standardized file formats. Thirdly, using interligation PETs (span ≥ 3 kb), loops are called and then annotated with peak support. Then, the binding peaks of the RNA of interest are identified using SPP or MACS2. The 2D chromatin interaction maps can be viewed in Juicebox.js. The loop file, peak file, coverage file, and domain file can be viewed in BASIC Browser.
ChIA-PET:Pair-end read (PET) sequences were scanned for the bridge linker sequence and only PETs with the bridge linker were used for downstream processing. After trimming the linkers, the sequences flanking the linker were mapped to the dm3 using bwa-mem and only uniquely aligned (MAPQ ≥ 30) PETs were retained. PCR duplicates were removed using Picard. Each PET was categorized as either a self-ligation PET (two ends of the same DNA fragment) or inter-ligation PET (two ends from two different DNA fragments in the same chromatin complex) by evaluating the genomic span between the two ends of a PET.
RNA-seq:the RNA-seq data was processed using STAR and RSEM. Firstly, the raw FASTQ files with pair-end reads were mapped to dm3 genome using STAR with ENCODE standard options. Secondly, FPKM of genes were calculated by utilizing ‘rsem-calculate-expression’ script of RSEM, with key configurations of ‘--paired-end’ and ‘--forward-prob 0’ for selecting reads that derived from the reverse strand. Then the uniquely mapped reads of total RNA-seq were hit and plotted the RPKM as boxplots.
Assembly: dm3
Supplementary files format and content: cluster, hic, bedgraph, peak
Library strategy: RDD
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| Submission date |
Apr 27, 2023 |
| Last update date |
May 21, 2024 |
| Contact name |
Tian (Simon) Zhongyuan |
| E-mail(s) |
simontian2020@outlook.com
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| Phone |
+86 15001976248
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| Organization name |
Southern University of Science and Technology (SUSTech)
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| Department |
School of Life Sciences
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| Lab |
Zheng Meizhen Lab
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| Street address |
1088 Xueyuan Avenue
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| City |
Shenzhen |
| State/province |
Guangdong |
| ZIP/Postal code |
518000 |
| Country |
China |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE207720 |
RDD reveals the landscape of RNA-associated chromatin DNA-DNA interactome and gene regulation |
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| Relations |
| BioSample |
SAMN34412628 |
| SRA |
SRX20121686 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7235320_RDD_roX2_Rep3_saturated_sequencing.50bp_tagextension.cluster.txt.gz |
3.0 Mb |
(ftp)(http) |
TXT |
| GSM7235320_RDD_roX2_Rep3_saturated_sequencing.cluster.txt.gz |
3.0 Mb |
(ftp)(http) |
TXT |
| GSM7235320_RDD_roX2_Rep3_saturated_sequencing.for.BROWSER.bedgraph.gz |
135.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM7235320_RDD_roX2_Rep3_saturated_sequencing.hic |
16.3 Mb |
(ftp)(http) |
HIC |
| GSM7235320_RDD_roX2_Rep3_saturated_sequencing.peaks.txt.gz |
196.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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