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Sample GSM4848678

Query DataSets for GSM4848678

Status

Public on Jan 26, 2021

Title

CRISPR_E2KO_LPS2h_Ccl2_pro_bait_rp3

Sample type

SRA

Source name

RAW264.7 cells

Organism

Mus musculus

Characteristics

cell type: Macrophage
strain: BALB/c
agent: LPS
time point: 2 hours

Treatment protocol

RAW264.7 cells were treated with/without LPS (2 hours)

Growth protocol

RAW264.7 cells were maintained normal 10% FBS DMEM medium

Extracted molecule

genomic DNA

Extraction protocol

WT or LNA control cells, CRISPR KO cells , Ccl2 E1 eRNA knockdown cells and GPS2/SMRT knockdown cells were re-suspended with 10 ml lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, for 10 minutes. Nuclei were re-suspended into 440 l Milli-Q water and 60 l of digestion buffer (15 l 10% SDS and 75 l 20% Triton X-100) was added. The cell lysis mixture was then incubated at 37 C for 2 hours in the shaker (900 RPM). 200U DpnII (NEB, R0543M) was added into the mixture three times. The lysis mixture was then incubated in the shaker overnight. The reaction mix was inactivated after overnight digestion by incubating at 65C for 20 min. The samples were then transferred to 50 ml Falcon tubes with 700 l ligation buffer and 5.5 ml Milli-Q water. The samples were then supplemented with 100 U T4 ligase (NEB, M0202M) and incubated overnight at room temperature. After that, samples were de-crosslinked at 65C for 12 hours. The ligated DNA was then purified by phenol-chloroform method, and further digested with NlaIII (NEB, R0125L) at 37 C overnight. The restriction enzyme was then inactivated for 20 min at 65 C. The DNA was further ligated at room temperature overnight. After the second ligation, the DNA was purified by QIAquick PCR purification kit
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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

NextSeq 550

Description

genomic DNA (bait PCR)
4C-seq_Sample

Data processing

Library strategy: 4C-seq
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.
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph files contain the genomic coordinates and p value of each peak.
ChIP-seq raw files (fastq, paired-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.
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph files contain the genomic coordinates and p value of each peak.
GRO-seq raw files (fastq, single-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using bwa 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.
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph files contain the genomic coordinates and p value of each peak.
4C-seq raw files (fastq, paired-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.
Genome_build: mm9
Supplementary_files_format_and_content: 4C-seq analysis does not provide processed data file.
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.
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph files contain the tags for each condense gene
ATAC-seq raw files (fastq, paired-end) were aligned to the NCBI37/mm9 version of the mouse reference genome, using Bowtie2 with default parameters. MACS2 was used for the peak calling. The differential changed peaks was done in R using edgeR.
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph files contain the genomic coordinates and p value of each peak.

Submission date

Oct 22, 2020

Last update date

Jan 26, 2021

Contact name

Zhiqiang Huang

E-mail(s)

zhiqiang.huang@ki.se, zhiqiang.huang@nju.edu.cn

Organization name

Nanjing University

Department

Medical School