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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 14, 2022 |
| Title |
107_cut_and_tag_shKDM1A_H3K9me3_rp2 |
| Sample type |
SRA |
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| Source name |
RAW 264.7
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| Organism |
Mus musculus |
| Characteristics |
cell type: Macrophage
strain: BALB/c
treatment: Control
chip antibody: H3K9me3
chip antibody vendor: Abcam
chip antibody cat.: ab8898
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| Treatment protocol |
ChIP-Seq:RAW264.7 cells were treated with or without LPS (100ng/ml) or IL4 (20ng/ml) treatment for indicated time.
ATAC-Seq: RAW264.7 cells were treated IL4 (20ng/ml) for 1h .
4C-Seq: RAW264.7 cells were treated with or without LPS (100ng/ml) or IL4 (20ng/ml) treatment for indicated time.
RNA-Seq: BMDM cells were treated with or without IL4 (20ng/ml) treatment for 6h.
Cut&Tag: No treatment
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| Growth protocol |
ChIP-Seq: WT RAW264.7 cells, lentivirus-medicated knockdown cells and GPS2 CRISPR KO cells were maintained normal 10% FBS DMEM medium.
ATAC-Seq: WT RAW264.7 cells were maintained normal 10% FBS DMEM medium.
4C-Seq: WT RAW264.7 cells were maintained normal 10% FBS DMEM medium.
RNA-Seq: BMDM cells were maintained normal 10% FBS DMEM medium supplement with 30% L929 medium for one week.
Cut&Tag: WT RAW264.7 cells were maintained normal 10% FBS DMEM medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-Seq: Fresh cells were crosslinked with 2 mM disuccinimidyl glutarate (DSG) for 30 min, followed by 1% formaldehyde for 10 min at room temperature. Nuclei were isolated, sonicated and incubated with magnetic bead-antibody complexes.
ChIP-Seq: ChIPed DNA was processed using Takara SMRTer ThruPLEX DNA-seq Kit. Briefly, DNA was end-repaired and ligated to stem-loop adaptors. Then DNA was PCR amplified with Illumina-compatible primers for 7-11 cycles and library fragments of 200 to 400 bp were selected using Ampure XP beads.
ATAC-Seq: WT RAW264.7 cells were seeded with triplicates in the 12 wells plates with 2×105 cells one day before the experiments. Cells were harvested with IL4 1h treatment, washed with PBS and then resuspended in lysis buffer. Cell nuclei were spined down and transferred into the transposition reaction buffer and incubated at 37 °C for 30 minutes using 105 cells. Genomic DNA was extracted using Qiagen MinElute PCR Purification Kit. ATAC-seq library was done with the protocol as previously reported (Buenrostro et al., 2015). The Purified ATAC-seq DNA library mix was sequenced on NextSeq 550 (Illumina, 35 SE reads, paired-end) in BEA Core Facility (Karolinska Institutet, Huddinge, Sweden).
ATAC-Seq: The ATAC-seq Library preparation was performed through the manufacturer's introduction.
4C-Seq: Fresh cells were crosslinked with 2% formaldehyde for 10 min at room temperature. Cells were counted and aliquoted with 10x7 cells per tube. For each 4C sample, 1 ml lysis buffer was applied for 10 min. Nuclei were spined down and discarded the supernatant. The nuclei were treated with the first digestion enzyme (Dpn II) overnight. The next day, the cell nuclei were heat-inactivated at 65C for 20 min and then treated with the first ligation enzyme overnight at room temperature. The genomic DNA was extracted and followed with the second digestion enzyme (Bfa I) overnight. Then heat-inactivated the enzyme and applied with then second the ligation enzyme overnight. The genomic DNA was purified again by phenol-chloroform extraction.
4C-Seq: PCR purified DNA (by baits) was processed using Takara SMRTer ThruPLEX DNA-seq Kit. Briefly, DNA was end-repaired and ligated to stem-loop adaptors. Then DNA was PCR amplified with Illumina-compatible primers for 7-11 cycles and library fragments of 200 to 400 bp were selected using Ampure XP beads.
RNA-Seq: 2×105 WT and GPS2 KO BMDM cells were seeded one day before the treatment. The next day the cells were treatment with IL4 6h and the total RNA was extracted from the manufacturer's introduction.
RNA-Seq: The RNA-seq library preparation was followed by the instructions of the NEB Next Ultra II RNA Library kit (NEB, E7770S).
Cut&Tag: 2×105 shGFP and shKDM1A RAW cells were seeded one day before.
Cut&Tag: The Cut and Tag Library preparation was performed through the manufacturer's introduction (Steven Henikoff lab,Bench top CUT&Tag V.3 ).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
library strategy: Cut&Tag
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| Data processing |
ChIP-seq raw files (fastq or txt files, single-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using Bowtie2 with default parameters. HOMER were then used for the tag directories construction from SAM files, duplicated reads removal, peak calling, peaks annotation, raw tag counts extraction and Bed file generation. Only peaks with fold enrichment over input tag count and local tag count > 4 and fdr< 0.001 (default -F -L -C -fdr) are kept.
Supplementary_files_format_and_content: ChIP-seq: Bedgraph files contain the genomic coordinates and p-value of each peak. The differential expression files were further provided.
ATAC-seq raw files (fastq, paired-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using Bowtie2 with default parameters. Peaks were called by MACS2. HOMER was then used for the tag directories construction from SAM files, duplicated reads removal, peak calling, peaks annotation, raw tag counts extraction and Bed file generation. Only peaks with fold enrichment over input tag count and local tag count > 4 and fdr< 0.001 (default -F -L -C -fdr) are kept.
Supplementary_files_format_and_content: ATAC-seq: Bedgraph files contain the genomic coordinates and p-value of each peak. The differential expression files were further provided.
4C-seq raw files (fastq, single-end) were performed by the standard 4Cseqpipe protocol. The primer sequences were trimmed from the raw reads. The trimmed reads were mapped to the mm9 reference fragmented genome. The contact intensity was normalized by the fragment counts. The cis-interacting DNA contact profiles around the baits were plotted in the same chromosome window. As the 4C-seq program only provides the heatmaps, only the list of the 4C primers which was used to amplified the signals is provided.
RNA-seq raw files (fastq, single-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using HISAT2 with default parameters. Read counts were imported by HOMER and analyzed using analyzeRepeats with the option rna and parameters -noadj -condenseGenes and -count exons for four replicates per condition. Differential gene expression was performed with DESeq2 using HOMER’s getDiffExpression.pl using default parameter. Transcripts with an adjusted p-value < 0.05 were considered as differentially expressed genes.
Supplementary_files_format_and_content: RNA-seq: Bedgraph files contain the genomic coordinates and p-value of each peak. The differential expression files were further provided.
Cut and Tag raw files (fastq, paired-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using Bowtie2 with default parameters. Peaks were called by MACS2. HOMER was then used for the tag directories construction from SAM files, duplicated reads removal, peak calling, peaks annotation, raw tag counts extraction and Bed file generation. Only peaks with fold enrichment over input tag count and local tag count > 4 and fdr< 0.001 (default -F -L -C -fdr) are kept.
Supplementary_files_format_and_content: CUT&TAG: Bedgraph files contain the genomic coordinates and p-value of each peak. The differential expression files were further provided.
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| Submission date |
Sep 27, 2021 |
| Last update date |
Dec 14, 2022 |
| Contact name |
Zhiqiang Huang |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
Nanjing University
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| Department |
Medical School
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| Street address |
Hankou 22
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210093 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE184884 |
GPS2 sensitizes IL4 pathway via the recruitments of KDM1A |
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| Relations |
| BioSample |
SAMN21873737 |
| SRA |
SRX12379791 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5599423_107_cut_and_tag_shKDM1A_H3K9me3_rp2.UCSC.bedGraph.gz |
53.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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