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| Status |
Public on Aug 22, 2021 |
| Title |
WYY35_HCP-3_Input_rep2 |
| Sample type |
SRA |
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| Source name |
Early embryos
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: WYY35
developmental stage: Early embryos
chip antibody: none
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| Growth protocol |
Synchronized Caenorhabditis elegans cultures were grown at 22 °C in batches of 500 ml using 2.8-L Fernbach flasks shaking at 230 rpm. Gravid adults were separated from debris by sucrose floating. Embryos were isolated by bleaching and fixed after chitinase treatment with 1 % formaldehyde for 10 min on ice.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as previously described (Gassmann et al., 2012) with slightly modification. Fixed embryos were suspended in 5 pellet volumes of ChIP buffer (+ protease inhibitors). 1 mL suspension was transferred to a milliTUBE (Covaris). Chromatin shearing was performed at 6-10 °C with Covaris M220 following settings: Processing Time: 8-20 min，Duty Cycle: 10%，Intensity: 75 Watts, Cycles per Burst: 200. The chromatin fragmentation was assessed by the smear pattern on 1% agarose gel, which was found to be between 200 and 500 bp. About 3 mg protein was diluted to 900 ml with ChIP buffer with 1 % Sarcosyl, 0.1 % Na-deoxycholate and 1 mM PMSF. 25 ml of the embryo lysate was used for the preparation of input DNA. 5 ml of antibodies was added (rabbit anti-HCP3, Novus Biologicals Q0804,1 mg/ml) to the extract and rotated gently at 4 °C overnight. 50 ml of dynabeads were added afterward and rotated at 4 °C for 2 hrs. Beads washing was performed using 2 X 1 ml FA buffer for 5 min, 1 ml FA-1000 for 10 min and 1 ml FA-500 buffer, successively. Beads were resuspended in 1 ml FA-500 buffer and transferred into new 1.5-ml Eppendorf tube, followed by 10 min FA-500 buffer washing, 10 min TEL buffer washing and brief washing by TE buffer. All washing steps were performed at 4 °C. Then, 50 ml of Elution buffer was added and incubated at 67 °C for 15 min. Dynabeads were separated from the eluted protein-DNA complex by a magnetic rack. The elutes were bought up to 150 ml by Elution buffer, followed by proteinase K digestion and reverse cross-linking at 65°C overnight. Both input DNA and ChIP-ed DNA were purified by ChIP DNA Clean & Concentrator Kit (Zymo research).
Library preparation and Illumina sequencing (Pair-End sequencing of 101 bp) were performed at the University of Hong Kong, Centre for Genomic Sciences (HKU, CGS). The 4 libraries (two replicates of Input and ChIP-ed DNA) were prepared based on the protocol of KAPA Hyper Prep Kit (KR0961 – v5.16). For each library, 0.8 ng of DNA was performed with reactions of end-repair, 3’ end A-tailing, and indexed adaptor ligation, followed by 16 cycles of PCR amplification reaction for library enrichment. After AMPure beads purification, each library was validated by Agilent Bioanalyzer, Qubit and qPCR for quality control analysis. The library was denatured and diluted to optimal concentration and applied in the cluster generation steps. HiSeq PE Cluster Kit v4 with cBot was used for cluster generation on the flow-cell. Illumina HiSeq SBS Kit v4 was used for Pair-End 101bp sequencing that runs on HiSeq 1500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
Input for WYY35_HCP-3_ChiP_rep2
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| Data processing |
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to WS245 whole genome using BWA-MEM (0.7.15)
Mapped reads from ChIP and input were used to call peaks and obtain read coverage per base using MACS version 1.4.2 under broad domain setting with P-value = 1e-3 cutoff.
Genome_build: WS245
Supplementary_files_format_and_content: bigwig
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| Submission date |
Feb 20, 2020 |
| Last update date |
Aug 22, 2021 |
| Contact name |
Zhongyang LIN |
| Organization name |
the University of Hong Kong
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| Street address |
4S17 Kadoorie Biological Sciences Building, Pokfulam Road
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| City |
Hong Kong |
| ZIP/Postal code |
na |
| Country |
China |
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| Platform ID |
GPL18730 |
| Series (1) |
| GSE145600 |
Formation of artificial chromosomes in Caenorhabditis elegans and analyses of their segregation in mitosis, DNA sequence composition and holocentromere organization |
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| Relations |
| BioSample |
SAMN14144206 |
| SRA |
SRX7756573 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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