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| Status |
Public on Jan 01, 2024 |
| Title |
Replicate 2 of G4P ChIP-seq in HCT116, input reads |
| Sample type |
SRA |
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| Source name |
colon cancer tissue
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| Organism |
Homo sapiens |
| Characteristics |
tissue: colon cancer tissue
cell line: HCT116
treatment: untreated
group: control group
target: G-quadruplex (G4P)
methodology: ChIP-seq
replicate: 2
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| Growth protocol |
Cells were grown in DMEM supplemented with 10% FBS and 1 × penicillin-streptomycin
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 0.5–1 × 108 transiently or stably transfected cells expressing G4P were crosslinked with 1% formaldehyde for 20 min at room temperature. Fixation was quenched by 0.125 M glycine for 15 min. The fixed cells were washed twice with PBS, suspended in NP-40 buffer (10mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% NP-40 and 2 mM AEBSF) and incubated on ice for 10 min. After centrifugation at 800×g for 5 min, the cell pellet was resuspended in a CHAPS buffer (20mM Tris-HCl pH 7.4, 0.5mMEGTA, 50 mM NaCl, 0.5% CHAPS, 10% glycerol and 2mM AEBSF). The suspension was incubated on ice for 30 min and centrifuged at 800×g for 5 min. The pellet was resuspended in 1 mL 1× dsDNase digestion buffer supplied with 50 μL dsDNase (Invitrogen, EN0771) and incubated at 37 ℃ for 20 min with constant agitation. A final concentration of 20 mM EDTA was added to terminate the reaction. The samples were pelleted by centrifugation at 15 000×g at 4 ℃ and the resulting supernatant was collected and incubated on ice. The pellet was resuspended in 500 μL wash buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.4, 0.1 mM EDTA, 0.5% Triton X-100) and sonicated for 30–60 s in an ice-cold water bath. After centrifugation at 15 000×g for 5 min, the supernatant of chromatin fragment was collected and combined with the supernatant from the previous step.
For library preparation, 50 μL of anti-FLAG M2 magnetic beads (Sigma-Aldrich) were washed with washing buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.5% Triton X-100) and blocked in the same buffer containing 75 μg/mL single-stranded sperm DNA and 1 mg/mL BSA. 1% chromatin fragment was saved as input and the remaining was incubated with blocked anti-FLAGmagnetic beads in rotation at 4 ℃ for 3 h. The beads were sequentially washed ten times with washing buffer and transferred to new tubes three times. The chromatin was eluted with 300 g/mL 3xFLAG peptide (Sigma-Aldrich) at 4 ℃ for 1 h. The eluted chromatin and the input sampleswere incubated with proteinase K at 65 ℃ overnight. After sequential RNase A and proteinase K digestion, DNA fragment was cleaned by extraction with phenol:chloroform:isoamyl alcohol, followed by ethanol precipitation. Libraries were constructed from the recovered DNA fragment using the NEBNext Ultra II DNA LibraryPrep Kit from Illumina (NEB) according to the manufacturer’s instructions. The next-generation sequencing was performed with Illumina HiSeq X Ten by Genewiz (Suzhou, China).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
We used fastp to preprocess our raw paired-end G4P ChIP-seq reads, which are allegedly trimmed. Fastp automatically filtered out low quality reads and detected adapter by complementing the paired sequences, which reported that all adapters were successfully trimmed beforehand (Command: --detect_adapter_for_pe). We also allowed it to correct low quality mismatch in the complement region of paired reads. (Command: --correction)
We utilized Bowtie 2 to align all G4P ChIP-seq reads to the primary assembly GRCh38 genome sequence downloaded from GENCODE, whose FM index was built by Bowtie 2 itself (Command: bowtie2-build). We used default parameters in the alignment process. The output “.sam” files were later converted to “.bam” format with SAMtools (Command: sort). These binary files were subsequently sent to BamTools for filtering out whose mapping quality was lower than 20 (Command: filter -mapQuality ">=20") and indexed with SAMtools (Command: index).
We entrusted deepTools to generate “.bigwig” files from “.bam” files. For G4P ChIP-seq data, we ignored all duplicative reads, and normalized the reads per genomic content (RPGC) to 1. We also extended these paired-end reads to match the fragment size. (Command: bamCoverage –ignoreDuplicates --normalizeUsing RPGC --extendReads)
Assembly: GRCh38
Supplementary files format and content: bigWig
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| Submission date |
Jul 31, 2023 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Ke-wei Zheng |
| E-mail(s) |
zhengkewei@hnu.edu.cn
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| Organization name |
Hunan University
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| Department |
School of Medical Sciences
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| Street address |
Lushan Road (S), Yuelu District
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| City |
Changsha |
| ZIP/Postal code |
410082 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (2) |
| GSE239692 |
G-quadruplex on Chromosomal DNA Negatively Regulates Topoisomerase 1 Activity [ChIP-seq] |
| GSE239694 |
G-quadruplex on Chromosomal DNA Negatively Regulates Topoisomerase 1 Activity |
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| Relations |
| BioSample |
SAMN36633315 |
| SRA |
SRX21187725 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7670069_HCT116_G4P_ChIP-seq_R2_Input.bigWig |
245.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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