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Sample GSM2643055

Query DataSets for GSM2643055

Status

Public on Jul 01, 2017

Title

ChIPseq_H3K27ac_2066_M_KO_neuronal

Sample type

SRA

Source name

adult mouse cortex

Organism

Mus musculus

Characteristics

antibody: H3K27ac
age: adult
gender: M
genotype: CK-KO
cell type: neuronal

Extracted molecule

genomic DNA

Extraction protocol

Nuclei were extracted from mouse brain tissue with or without formaline fixation. Neuronal nuclei were collected by FACS. Cromatin was fragmented by Mnase digestion (histone ChIP) or sonication (CTCF ChIP), and immunoprecipitated by different antibodies.
ChIP DNA was end repaired (End-it DNA Repair kit; Epicentre) and A tailed (Klenow Exo-minus; Epicentre). Adaptors (Illumina) were ligated to the ChIP-DNA (Fast-Link kit; Epicentre) and then PCR amplified using Illumina TruSeq ChIP Library Prep Kit. Library DNA with expected size (NChIP, ~275bp; XChIP, 350bp to 500bp) was selected by Pippin.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

ChIPseq_H3K27ac_neuronal_KOvsWT_1kb_001_annotated.txt

Data processing

RNA-seq: The sequenced RNAseq data were aligned to the Mouse (mm10) reference genome using Tophat2 short-read aligner. Reads were counted using HTSeq against the Gencode vM4 Mouse annotation. Genes were filtered based on the criteria that all replicates in either condition must have at least 5 reads per gene. On the resulting filtered transcript, a pairwise differential analysis between Setdb1 conditional mutant vs control cortex was performed using the voom-limma R package, which converts counts into precision weighted log2 counts per million and determines differentially expressed genes using a linear model. Significantly differentially expressed genes were identified using a cutoff of Benjamini-Hochberg adjusted p-value less than 0.05.
ChIP-seq: The sequenced ChIP-seq data was first checked for quality using the various metrics generated by FastQC (v0.11.2). Raw sequencing reads were then aligned to the Mouse (mm10) reference genome using default settings of Bowtie (v2.2.0). Uniquely mapped reads were retained and the alignments were subsequently filtered using the SAMtools package (v0.1.19) to remove duplicate reads. Differential analysis between mutant and control samples was performed using diffReps with window size 1000 bp and moving step size 500 bp, and FDR<5% as significance cutoff.
insituHiC: The processing of the raw 2x125bp read pair in-situ HiC FASTQ files was performed using the HiC-Pro analysis pipeline. In brief, HiC-Pro performs four major tasks: aligning short reads, filtering for valid pairs, binning, and normalizing contact matrices. HiC-Pro implements the truncation-based alignment strategy using Bowtie v2.2.318, mapping full reads end-to-end or the 5’ portion of reads preceding a GATCGATC ligation site that results from restriction enzyme digestion with MboI followed by end ligation. Invalid interactions such as same-strand, dangling-end, self-cycle, and single-end pairs are not retained. Binning was performed in 20kb non-overlapping, adjacent windows across the genome and resulting interaction matrices were obtained.
Genome_build: mm10
Supplementary_files_format_and_content: text files of differential expression genes (RNAseq), differential binding regions (ChIPseq) and interaction counts (insituHiC)
Supplementary_files_format_and_content: ChIPseq_CTCF_neuron_KO_vs_WT_1kb_001b.annotated.txt: text file of differential binding regions
Supplementary_files_format_and_content: ChIPseq_H3K27ac_neuron_KO_vs_WT_1kb_001a.annotated.txt: text file of differential binding regions
Supplementary_files_format_and_content: ChIPseq_H3K9me3_neuron_KO_vs_WT_1kb_001a.annotated.txt: text file of differential binding regions
Supplementary_files_format_and_content: RNAseq_Setdb1-CK-KO_KO_vs_WT_gencode_vM4.txt: text file of differential expression genes
Supplementary_files_format_and_content: insituHiC_432_M_WT_neuronal_20000.HiCPro.interaction.txt: text file of interaction raw counts
Supplementary_files_format_and_content: insituHiC_513_M_CK-KO_neuronal_20000.HiCPro.interaction.txt: text file of interaction raw counts
Supplementary_files_format_and_content: insituHiC_9836_M_WT_neuronal_20000.HiCPro.interaction.txt: text file of interaction raw counts
Supplementary_files_format_and_content: insituHiC_9837_M_CK-KO_neuronal_20000.HiCPro.interaction.txt: text file of interaction raw counts
Supplementary_files_format_and_content: insituHiC_20000.HiCPro.bed: bed file of interaction bins

Submission date

May 26, 2017

Last update date

May 15, 2019

Contact name

Eddie Loh

Organization name

Icahn School of Medicine at Mount Sinai

Department

Department of Neuroscience

Street address

1425 Madison Avenue