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| Status |
Public on Mar 03, 2021 |
| Title |
Prdm16_mutant_FB_ATAC-seq_Rep2 |
| Sample type |
SRA |
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| Source name |
forebrain
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| Organism |
Mus musculus |
| Characteristics |
tissue: forebrain
genotype: homozygous mutant
developmental stage: E13.5
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Forebrain from E13.5 wt and mut embryos were collected, nuclei were extracted according to published protocol (Buenrostro et al. 2013).
The ATAC-seq libraries were made according to the published method (Buenrostro et al. 2013) and using the Illumina Nextera DNA library kit. In brief, cortices were dissected from 3 control and 3 Prdm16 KO E13.5 brains. Tn5 enzyme reaction was performed at 37 degrees for 30mins, followed by DNA purification. 11 cycles of PCR amplification was performed using barcoded adaptors and primers on purified DNA template. Libraries were purified and pooled before sequencing with illumina Next-seq platform.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Prdm16_mutant_FB_ATAC-seq_Rep2
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| Data processing |
ATAC-Seq: The replicated Prdm16 KO (x3) and control (x3) ATAC-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the UCSC Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters. The high quality and uniquely mapped reads (with mapping quality >= 20) were used for further analysis. ATAC-seq differential expression analysis between Prdm16 mutants and wild types on the Prdm16 bound ChIP-seq peaks were performed by Limma R package. The ATAC-seq peak calling was performed by HOMER using the “broad peak” option with parameters “-region -size 1000 -minDist 2500”, separately for the mutant and wild type. To compare active enhancers between E13.5 and E15.5, we further re-analyzed the publicly available histone mark H3K27ac and PRDM16 ChIP-seq data at E15.5. We called the peaks against Input using “narrow peak” option by HOMER with the default parameters.
RNA-Seq: After trimming the adaptor sequences using Trimmomatic, we mapped RNA-seq reads from the replicated Prdm16 wild type (x3) and mutant samples (x3) to the UCSC Mus musculus (mm10) genome assembly using HISAT2. We normalized RNA-seq by the “Relative Log Expression” method implemented in the DESeq2 Bioconductor library (Love et al., 2014). Gene annotation was obtained from the iGenomes UCSC Mus musculus gene annotation. Differentially expressed mRNAs between Prdm16 mutants versus wild type were identified, and FDR (Benjamini-Hochberg) was estimated, using DESeq2.
ChIP-Seq: The replicated Prdm16 KO (x3) and control (x3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the UCSC Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters. The uniquely mapped reads (with mapping quality >= 20) were used for further analysis. The PRDM16 peaks were called by HOMER (v4.10) (Heinz et al., 2010). The peak replicate reproducibility was estimated by Irreproducibility Discovery Rate (IDR), using the HOMER IDR pipeline (https://github.com/karmel/homer-idr). As suggested by the Encode IDR guideline that IDR requires to initially call peaks permissively for the replicates, we used a relatively relaxed parameter “-F 2 -fdr 0.3 -P .1 -L 3 -LP .1” for the true/pseudo/pooled replicates by the HOMER peak calling. The final confident peaks were determined by an IDR < 5%. The peaks that were overlapped with mm10 blacklist were also removed.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig for coverage; xlsx file for RNA-seq count table
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| Submission date |
Dec 08, 2020 |
| Last update date |
Mar 05, 2021 |
| Contact name |
Jiayu Wen |
| Organization name |
John Curtin School of Medical Research, The Australian National University
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| Department |
Department of Genome Science
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| Lab |
The Wen Lab
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| Street address |
133 Garran Rd
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| City |
CANBERRA |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE162854 |
PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors |
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| Relations |
| BioSample |
SAMN17028656 |
| SRA |
SRX9651582 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4963015_FBmutant2_q20.mm_norm.bw |
526.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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