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| Status |
Public on Mar 07, 2019 |
| Title |
Wt2 Kat2b promoter 4Cseq |
| Sample type |
SRA |
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| Source name |
Wild type cultured fP0 forebrain NSC
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6/DBA
genotype/variation: Wild type
cell type: forebrain-derived neural stem cells
passages: passage 3 to 7
viewpoint: Kat2b promoter
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| Treatment protocol |
Cells from individual brain cultures were fixed with 1% formaldehyde solution in 10 million cells aliquots for subsequent 4Cseq analysis. Briefly, cells from each individual mouse culture were crosslinked with 1% formaldehyde and snap-frozen in 10 million cells aliquots. Cells (one aliquot) were then lysed with the lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100 and 1 x complete protease inhibitor) using a douncer. The collected nuclei were digested with 600 U of the first enzyme followed by ligation with 100 U T4 DNA ligase (Roche). 600 mg Proteinase K (Invitrogen) and 300 mg RNase A (Invitrogen) were added and incubated sequentially, respectively. Following the phenol-chloroform purification, the samples were treated with a second round of digestion using the second enzyme and self-ligated with 200 U T4 DNA ligase (Roche). The phenol-chloroform purified self-ligation products were used as templates for 4C-PCR. 200 μl PCR reactions using 500 ng of 4C template were performed for 28 cycles for each viewpoint and pooled together. The PCR products were purified using AMPure beads (Beckman-Coulter) according to the manufacturer’s instructions and pooled for sequencing on the Illumina NextSeq 500.
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| Growth protocol |
P0 brain-derived NSC cultures were obtained from dissected forebrain of wild type and Sox2-deleted P0 mice, and grown, as described in (Favaro et al., Nat. Neurosci. 2009, 12:1248-56) and (Zhang et al., Nature 2013). Cells were grown in parallel for two passages in 10 ng/ml Epidermal Growth Factor (EGF) and 10 ng/ml basic Fibroblast Growth Factor (bFGF), followed by cell dissociation and expansion in the presence of EGF, but not bFGF, for 3-4 more passages, as described in Zhang et al. (2013), for optimal maintenance of mutant NSC (Favaro et al., 2009). The culture of Sox2-deleted NSC was performed in parallel with that of wild type NSC (Zhang Y et al., Nature 2013, 504:306-10). The background of mice was C57BL/6/DBA (as in Favaro et al., 2009).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The extraction procedures were performed as described in Stadhouders et al., 2013.
4C-seq libraries were prepared as described in Stadhouders et al., 2013. Briefly, nine regions of seven genes were chosen as viewpoints based on their relevance to brain development, presence of interaction detected by the ChIA-PET analysis and observed differential connectivity between wild type and mutant cells. The primers were designed using Primer Designer for 4C Viewpoints (https://mnlab.uchicago.edu/4Cpd/). Depending on the locus of interest, we used one of the three different 4 base cutters, DpnII (NEB), Csp6I (Thermo Scientific) or NlaIII (NEB), for the first or second rounds of restriction enzyme digestion. The primer sequences, viewpoint regions and the restriction enzyme used for each viewpoint were listed in Table S11 of the publication. Briefly, ten million cells from each individual mouse were crosslinked with 1% formaldehyde and lysed with the lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100 and 1 x complete protease inhibitor) using a douncer. The collected nuclei were digested with 600 U of the first enzyme followed by ligation with 100 U T4 DNA ligase (Roche). 600 mg Proteinase K (Invitrogen) and 300 mg RNase A (Invitrogen) were added and incubated sequentially, respectively. Following the phenol-chloroform purification, the samples were treated with a second round of digestion using the second enzyme and self-ligated with 200 U T4 DNA ligase (Roche). The phenol-chloroform purified self-ligation products were used as templates for 4C-PCR. 200 μl PCR reactions using 500 ng of 4C template were performed for 28 cycles for each viewpoint and pooled together. The PCR products were purified using AMPure beads (Beckman-Coulter) according to the manufacturer’s instructions and pooled for sequencing on the Illumina NextSeq 500.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Restriction enyzmes: RE1 DpnII, RE2 Csp6I
Viewpoint: chr17: 53707484-53707872
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| Data processing |
Library strategy: 4C-seq
BCL2fastq version 2.18.0.12 was used to demultiplex data and convert BCL files generated by Illumina (Nextseq) system to fastq files.
Sequenced reads were trimmed for view-point specific primer sequence (reverse complement), and masked for low-quality calls with Cutadapt version 1.8.3; parameters: -q 30 --trim-n -e 0.1 --minimum-length 15.
4cker pipeline is used to process the reads. First, reduced genome reference files containing reference sequence flanking RE1 sites were made for specific RE1 and read length (following https://github.com/rr1859/R.4Cker). Second, bowtie index files were then built. Third, the reads were aligned to the reduced genome using bowtie2 with default parameters. Software: bedtools, oligoMatch from kent tools, and bowtie2.
BedGraph generation: the mapped reads, which contain restriction enzyme (RE) fragments, were quantified similar to 4cker suggestion (cat mapped.sam| grep -v "^@" | awk '$5 > 39 {print $3}'| grep "^chr" | sort | uniq -c | sed -e 's/_\|:\| \|-/\t/g' | awk '{print $2"\t"$3"\t"$4"\t"$1 }').
Analysis was performed with 4cker library in R: library(R.4Cker) and the normalized 4C count bedGraph files from cisAnalysis (parameter k=10) were generated and deposited here.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: bedGraph files
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| Submission date |
Feb 27, 2018 |
| Last update date |
Mar 07, 2019 |
| Contact name |
Federico Zambelli |
| Organization name |
University of Milan
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| Department |
Department of Biosciences
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| Street address |
Via Giovanni Celoria 26
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| City |
Milano |
| ZIP/Postal code |
20133 |
| Country |
Italy |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE90561 |
Sox2 is required for functional chromatin connectivity in brain-derived neural stem cells. |
| GSE111190 |
4Cseq analysis of neural stem/progenitor cells (NSC) from wild type and Sox2-deleted mouse neonatal forebrain |
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| Relations |
| BioSample |
SAMN08616967 |
| SRA |
SRX3746726 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3025064_Kat2b.Wt2_cis_norm_counts.bedGraph.gz |
21.3 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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