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Sample GSM5024313 Query DataSets for GSM5024313
Status Public on Jan 18, 2021
Title Dam-Only Rep 3
Sample type SRA
Source name E17.5 Cortex
Organism Mus musculus
Characteristics strain: MF1
tissue: Cortex
developmental stage: E17.5
treatment: Dam-Only Construct
Treatment protocol CHD8 TaDa (0.5ug/ul) or Dam-only (0.5ug/ul) and electroporation-control (0.25ug/ul) plasmids were injected into the fetal brain ventricles at embryonic day (E) 13.5 before collection at E17.5.
Extracted molecule genomic DNA
Extraction protocol Sample genomic DNA extraction was performed using the Qiagen QIAamp DNA Micro Kit (Qiagen, 56304). Extracted genomic DNA was digested overnight at 37°C with DpnI (NEB, R0176S) to cut adenine-methylated GATC sites. Following digestion, DNA was column purified with the QIAquick PCR Purification Kit (Qiagen, 28104) to remove un-cut genomic DNA. dsADR adaptors (0.05 umole, DST purity, Sigma) were blunt-end ligated to DpnI-digested fragments using T4 DNA ligase (NEB, M0202S; 2 hours at 16°C, heat inactivation at 65°C for 20 minutes) to prepare for PCR amplification. Before PCR amplification, fragments were digested with DpnII (NEB, R0543S) to cut non-methylated GATC sites and prevent amplification of unmethylated regions and purified with a 1:1.5 ratio of Seramag beads (Fisher Scientific, 12326433). PCR amplification of DpnII-digested fragments using MyTaq (Bioline, BIO-21112) enriched for methylated fragments before samples were sonicated and prepped for sequencing. Sonicated samples were subjected to AlwI digestion (NEB, R0513S) to remove previously ligated adaptors and initial GATC sequences from fragments.
A modified TruSeq protocol was used to generate sequencing libraries involving end repair, 3’ end adenylation, sequencing adaptor ligation (0.05 umole, DST purity, Sigma), and DNA fragment enrichment using a reduced number of PCR cycles. TaDa-seq libraries were sequenced on the Illumina HiSeq 1500 platform using a 50bp strategy by the Gurdon Institute Next Generation Sequencing Core.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1500
Data processing Library strategy: DamID-seq
Unaligned TaDa-seq reads were trimmed using TrimGalore (Version 0.4.2), assessed for general quality control with the FastQC tool (Version 0.11.9), and aligned to the mouse reference genome (mm10) using BWA (Version 0.7.17). Biological replicates were analyzed independently and as a single merged file generated via samtools (Version 1.10).
TaDa-seq peak calling was performed using MACS2 (Version 2.2.5) with model-based peak identification disabled, a p-value cutoff set at less than 0.00001, and the merged Dam-only dataset as a control. Peak calling for individual CHD8 TaDa replicates was performed against the merged Dam-only dataset to identify specific peaks that were enriched in CHD8 TaDa versus non-specific signal in Dam-only experiments. Peak calling for the Dam-only merged dataset was performed without a control dataset as Dam-only is analogous to assays of accessible chromatin. A final set of merged CHD8 TaDa peaks was obtained using bedtools intersect (Version 2.29.2) to select peaks that were present in at least 3 replicates and had a MACS2 FDR less than 0.00001.
Enriched regions from TaDa-seq datasets were annotated to genomic features using custom R scripts and combined UCSC and RefSeq transcript sets. CHD8 target genes were assigned to nearest transcription start site, which for distal peaks was achieved using the bedtools closest command (Version 2.29.2). Bigwig coverage files were generated using deeptools bamCoverage (Version 3.3.1).
Genome-wide signal summary correlation heatmaps were generated using the multiBigwigSummary and plotCorrelation tools from deeptools (Version 3.3.1). Peak loci heatmaps were generated using the deeptools computeMatrix and plotHeatmap tools (Version 3.3.1). Analysis of peaks not captured by the CHD8 TaDa experiments was performed using bedtools intersect (Version 2.29.2) with CHD8 TaDa filtered peak datasets. Promoter-proximal versus promoter-distal and peak set concordance datasets were also obtained using the bedtools intersect tool (Version 2.29.2). Ontology analysis was performed using the GREAT online tool (Version 4.0.4) or goseq (Version 1.36.0). HOMER was used to perform de novo motif discovery with default parameters (Version 4.10).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: broadPeak or bed peak files
Supplementary_files_format_and_content: bigWig coverage files
Submission date Jan 17, 2021
Last update date Jan 19, 2021
Contact name Alex Nord
Phone 530-754-5022
Organization name University of California, Davis
Department Neurobiology, Physiology, & Behavior
Street address 1544 Newton Court
City Davis
State/province CA
ZIP/Postal code 95618
Country USA
Platform ID GPL18480
Series (1)
GSE165002 Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID
BioSample SAMN17369179
SRA SRX9864019

Supplementary file Size Download File type/resource 87.1 Mb (ftp)(http) BW
GSM5024313_Dam-pCAG-iue-77-3_S3.ext300.no_model_peaks.broadPeak.gz 1.5 Mb (ftp)(http) BROADPEAK
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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