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| Status |
Public on Mar 20, 2023 |
| Title |
THP-1_CTRL-input |
| Sample type |
SRA |
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| Source name |
THP-1_CTRL-input
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| Organism |
Homo sapiens |
| Characteristics |
cell line: THP-1
cell type: AML cell line
treatment: DMSO for 48hrs
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| Treatment protocol |
THP-1 cells were treated with 2 μM OPA1 or DMSO control for 48 hrs.
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| Growth protocol |
THP-1 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA), supplemented with 10% FBS (Gibco) and penicillin-streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was obtained from 5x10^6 cells with Cell/Tissue DNA Isolation Mini Kit (Vazyme)
50 ng genomic DNA extracted from THP-1 cell was fragmented into 300 bp reads by use of Ultrasonic Bioruptor sonicator (Diagenode Bioruptor). Then, the selective 5hmC chemical labeling was performed. The above fragmented DNA was added into 60 μl reaction buffer containing 6 μl of 10×β-GT reaction buffer, 2 μl of N3-UDP-Glc (3 mM), and 3 μl of β-GT (40 mM), and incubated at 37°C for 2 hr. The reaction product was purified with magnetic beads and DNA was eluted with 60 μl ddH2O. DMCO-S-S-PEG-Biotin (1 mM) was added into the DNA solution to reach a final concentration as 150 μM, and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by 50 µl Dynabeads MyOne Streptavidin C1 beads (Life Technologies) for 30 min at room temperature. Next, the captured DNA fragments were subjected to 18 cycles of PCR amplification using Genomic DNA Sample Prep Kit (Illumina). The input library was prepared from fragmented DNA through direct PCR, without chemical labeling or capture. The libraries were denatured with 0.1 M NaOH to generate single-strand DNA (ssDNA) molecules and were added to the flow cell channel at a concentration of 8 pM. The libraries were quantified with a Qubit fluorometer (Life Technologies) and sequenced on a HiSEQ4000 sequencer (Illumina) with paired-end 50-bp reads.
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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: 5hmC-Seal
Basecalls performed using HiSEQ4000 sequencer (Illumina)
Off-Line Basecaller (OLB V1.8) was used to recognize the image files generated by sequencing to obtain sequence files, and the data was preliminarily filtered by Solexa CHASTITY. Data quality was identified using FastQC (v0.11.7).
5hmC reads were deeper filtered with Trimmomatic and were mapped to Ensembl GRCh37 human reference genome using HISAT2. The identification of 5hmC peaks in each sample was performed using MACS v249, and an IDR cutoff of 0.05 was used to filter high confident peaks.
DiffBind was used to determine the differences in 5hmC density according to the peak numbers at the same gene locus of different samples.
5hmC-seq reads were aligned to the hg19 genome assembly using IGV_2.8.0
Genome_build: hg19
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| Submission date |
Mar 25, 2020 |
| Last update date |
Mar 20, 2023 |
| Contact name |
Jun Lu |
| E-mail(s) |
lujun4726@zju.edu.cn
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| Phone |
18258826247
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| Organization name |
Zhejiang University
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| Street address |
866 Yuhangtang Road
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
3100058 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE147509 |
Opioid receptor signaling suppresses leukemia by inducing TET-dependent DNA demethylation [5hmC-seq] |
| GSE147511 |
Opioid agonists as potential AML therapy |
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| Relations |
| BioSample |
SAMN14446502 |
| SRA |
SRX7992103 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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