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| Status |
Public on May 19, 2022 |
| Title |
RKO_shZNF280C_H3K27me3-rep2 |
| Sample type |
SRA |
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| Source name |
RKO colon cancer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: RKO
genotype/variation: stably expressing a ZNF280C-targeting shRNA controlled by a tetracycline-inducible promoter
chip antibody: SMCHD1
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| Treatment protocol |
RKO colon cancer cells stably expressing a ZNF280C-targeting shRNA controlled by a tetracycline-inducible promoter were treated with or without 1 ug/ml doxycycline for 72 hours, and the cells were harvested for analysis as described below.
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| Growth protocol |
RKO cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked in 1% formaldehyde for 10 min at room temperature (RT) with gentle swirling, and crosslinking was stopped by adding glycine to a final concentration of 0.125 M and incubating at RT for 5 min. Cells were harvested and an aliquot of 1 × 10^7 cells were used per ChIP reaction. Each cell aliquot was resuspended in 0.5 mL of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4), and chromatin was fragmented on ice to 200 to 500 bp in size with 5 pulses of 30 sec at 50% power with a Sonics VibraCell sonicator. 5-10 μg primary antibodies or control IgG were pre-conjugated with Protein G magnetic beads (Pierce) at 4°C overnight. 1% of sheared chromatin was saved as input, and the rest was incubated with pre-conjugated antibody on a rotator at 4°C overnight. The beads were washed 5 times with LiCl wash buffer (0.1 M Tris, 0.5 M LiCl, 1% NP-40, 1% sodium deoxycholate, pH 7.5) followed by a single wash with TE buffer (10 mM Tris-HCl, 0.1 mM Na2EDTA, pH 7.5). The beads were then resuspended in 200 μL IP elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65°C for 1 hour in a heat block with vortexing. The supernant was collected and together with input was incubated at 65°C for 16 hours to reverse cross-links. DNA was purified with QIAquick PCR Purification Kit (Qiagen) and eluted in 50 μL EB.
ChIP’s DNA was subjected to end-repair and then was 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated fragments. Fragments were amplified by PCR and PCR products were purified and selected with the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Library was qualified by Qubit ssDNA kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
MGISEQ-2000RS |
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| Data processing |
Raw reads were aligned to hg19 using bwa. The sam files were converted to bam with samtools.
MACS2 was used to call peaks on the bam files with signals from input DNA as reference.
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files were generated using bamCoverage from Deeptools.
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| Submission date |
Mar 07, 2022 |
| Last update date |
May 19, 2022 |
| Contact name |
Xingsheng Shu |
| E-mail(s) |
shuxingsheng@gmail.com
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| Organization name |
Shenzhen University
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| Department |
Health Science Center
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| Street address |
No 3688, Nanhai Avenue
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| City |
Shenzhen |
| State/province |
Guangdong |
| ZIP/Postal code |
518060 |
| Country |
China |
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| Platform ID |
GPL30209 |
| Series (2) |
| GSE181855 |
Epigenomic and transcriptomic features induced by silencing of ZNF280C in colon cancer cells and knock-out of Zfp280c in mice [ChIP-Seq, ATAC-Seq] |
| GSE181856 |
Zinc finger protein 280C contributes to colorectal tumorigenesis by maintaining epigenetic repression at H3K27me3-marked loci |
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| Relations |
| BioSample |
SAMN26514134 |
| SRA |
SRX14406888 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5939233_RKO_shZNF280C_H3K27me3-rep2.bw |
652.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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