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Sample GSM5599390 Query DataSets for GSM5599390
Status Public on Dec 14, 2022
Title 74_RAW_CRISPR_GPS2_KO_H3K4me1_rp2
Sample type SRA
 
Source name RAW 264.7
Organism Mus musculus
Characteristics cell type: Macrophage
strain: BALB/c
treatment: Control
chip antibody: H3K4me1
chip antibody vendor: Abcam
chip antibody cat.: ab176877
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
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.
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 ).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model NextSeq 550
 
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.
 
Submission date Sep 27, 2021
Last update date Dec 14, 2022
Contact name Zhiqiang Huang
E-mail(s) [email protected], [email protected]
Organization name Nanjing University
Department Medical School
Street address Hankou 22
City Nanjing
State/province Jiangsu
ZIP/Postal code 210093
Country China
 
Platform ID GPL21626
Series (1)
GSE184884 GPS2 sensitizes IL4 pathway via the recruitments of KDM1A
Relations
BioSample SAMN21873754
SRA SRX12379758

Supplementary file Size Download File type/resource
GSM5599390_74_RAW_CRISRP_GPS2_KO_H3K4me1_rp2.UCSC.bedGraph.gz 114.1 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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