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Sample GSM2401618

Query DataSets for GSM2401618

Status

Public on Jan 01, 2017

Title

KO2_ATAC

Sample type

SRA

Source name

Chd7-deficient_GCps_ATAC-seq

Organism

Mus musculus

Characteristics

strain background: C57Bl/6J
age: P7
genotype/variation: Chd7-deficient
genotype/variation: Math1cre;Chd7flox/flox
cell type: cerebellar granule cell precursors (GCps)

Extracted molecule

genomic DNA

Extraction protocol

ATAC-seq: Protocol as described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, with the excpetion that the Nuclei EZ Prep Nuclei Isolation Kit (Sigma, NUC-101) was used for nuclei isolation.
ATAC-seq: Protocol as described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, using illumina barcoded primers for sequencing.
RNA-seq: The cerebella of post natal day 7 Chd7f/f (cn) and Math1cre;Chd7f/f (cko) pups were dissected in DPBS (Gibco) and lobules IX and X were removed. The cerebella of the individuals from each genotype were pooled. Cells were dissociated using Papain I (100U, Worthington) followed by trituration. Granule cell precursors were isolated by Percoll fractionation with a 35% and 65% Percoll gradient (Sigma) as described (Yu T, Meiners LC, Danielsen K, Wong MT, Bowler T, Reinberg D, Scambler PJ, van Ravenswaaij-Arts CM, and Basson MA. Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome. eLife. 2013;2(e01305.). Total RNA was extracted from freshly isolated cells (5x106/cerebellum) with an Agilent Absolutely RNA miniprep kit (Agilent Technologies, Stockport, UK)
RNA-seq: RNA quality and integrity was assessed on a 2100 Bioanalyzer and 2200 Tapestation using an R6K screenTape (Agilent Technologies, Stockport, UK). After quantification on Qubit using Quant-iT RNA assay kit (Life Technologies, Paisley, UK), 2g total RNA was used from each sample for library preparation using an Illumina TruSeq kit (Illumina, Cambridge, UK). Libraries were resolved by agarose gel electrophoresis, 250-550bp DNA fragments isolated and gel purified with a Qiagen DNA gel extraction kit (Qiagen, Manchester, UK). Two libraries with unique barcodes were combined and sequenced per lane on a HiSeq2000 (Illumina, Cambridge, UK) at the Biomedical Research Centre, King’s Health Partners.
ChIP-seq: The cerebella of post natal day 7 Chd7f/f (cn) and Math1cre;Chd7f/f (cko) pups were dissected in DPBS (Gibco) and lobules IX and X were removed. The cerebella of the individuals from each genotype were pooled. Cells were dissociated using Papain I (100U, Worthington) followed by trituration. Granule cell precursors were isolated by Percoll fractionation with a 35% and 65% Percoll gradient (Sigma) as described (Yu T, Meiners LC, Danielsen K, Wong MT, Bowler T, Reinberg D, Scambler PJ, van Ravenswaaij-Arts CM, and Basson MA. Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome. eLife. 2013;2(e01305.).
ChIP-seq: Freshly isolated cells (2-3x107) of each genotype were fixed, chromatin isolated, precipitated with an antibody to H3K4me1 (Abcam, Cambridge UK) and ChIP-seq libraries produced and sequenced as previously described (Tee WW, Shen SS, Oksuz O, Narendra V, and Reinberg D. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell. 2014;156(4):678-90.).

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

processed data file
master_sheet_atac_all_datav2.xlsx

Data processing

ATAC-seq:
15bp was trimmed from the raw sequencing reads using trim_galore [version 0.4.1] with parameters as follows: trim_galore --nextera --paired --quality 20 --stringency 5 --gzip --clip_R1 15 --clip_R2 15 --length 25 -o [output_filename]
Reads were uniquely aligned to the mouse reference genome (mm10) using Bowtie [version 1.1.1] with a maximum insert size for valid paired-end alignments of 2000 and a maximum number of attempts of 200 to match an alignment for one mate with an alignment for the opposite mate.
bam files were sorted and indexed using samtools [version 0.1.19-96b5f2294a]
Peaks were called in three WT replicates by comparing them to three KO samples and vice versa using MultiGPS [version 0.5] with the minimum q-value for reported peaks set at 0.05 and the mitochondrial genome excluded.
The bedtools window function from BEDTools [version 2.17.0] was used to identify ATAC-seq peaks within 1000bp of DEGs, and Dnase-seq peaks within 100bp of ATAC-seq peaks.
RNA-seq:
RNAseq reads were aligned to the mouse reference genome (mm10) using TopHat [version 2.0.14] with an expected inner distance between mate pairs of 150bp and standard deviation for the distribution on inner distances between mate pairs of 50bp. The transcriptome index was created using ENSEMBL build 75
HTseq [version 0.6.1p1] was used to estimate the number of RNA sequencing reads supporting the expression of protein coding genes. For this, an intersection-strict mode was used and all reads with alignment quality lower than 3 skipped.
Differential expression was analyzed with DESeq2 1.8.1. Genes with an FDR<0.05 were considered differentially expressed.
ChIP-seq:
Reads were aligned to the mouse reference genome (mm10) using Bowtie [version 1.1.0] , only allowing uniquely mapping reads, with an insert size of 1000 bases. Default values were used for all other parameters
Following mapping, PCR duplicates were removed using the samtools 'rmdup' command
Identification of enriched regions was performed by comparing the ChIP to the input reads using macs2 [2.1.0.20140616], with a p-value cutoff of 0.0001 and 3,90 as the model building parameters. The bedGraph output generated by macs2 was used to visualise the ChIP and input signals.
Genome_build: mm10
Supplementary_files_format_and_content: master_sheet_atac_all_datav2.xlsx: Excel document with sheets containing coordinates of all identified peaks,i.e. CTRL Vs. KO peaks KO Vs. CTRL peaks All ATAC peaks All DNase Peaks CTRL Vs. KO peaks within 1kb of a downregulated gene (incl. names of all unique genes) CTRL Vs. KO peaks within 1kb of a upregulated gene (incl. names of all unique genes) KO Vs. CTRL peaks within 1kb of a downregulated gene (incl. names of all unique genes) KO Vs. CTRL peaks within 1kb of a upregulated gene (incl. names of all unique genes)
Supplementary_files_format_and_content: DEGs_Chd7.xls Excel file containing all differentially expressed genes as identified by DESeq2, including log2 fold change values and FDR.
Supplementary_files_format_and_content: WT_R1_htseq_strict_a3_s_counts.txt WT_R2_htseq_strict_a3_s_counts.txt KO_R1_htseq_strict_a3_s_counts.txt KO_R2_htseq_strict_a3_s_counts.txt → All text files containing raw read counts for the corresponding fastq file
Supplementary_files_format_and_content: H3K4me1_WT.bw; Signal profile file showing the extent of H3K4me1-DNA binding genome wide

Submission date

Nov 23, 2016

Last update date

May 15, 2019

Contact name

Albert Basson

E-mail(s)

albert.basson@kcl.ac.uk

Organization name

King's College London

Department

Craniofacial Development and Stem Cell Biology