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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 01, 2023 |
| Title |
ARMC5_WT_NSC 2 |
| Sample type |
SRA |
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| Source name |
Neural stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Neural stem cells
strain: 129 sv X CD1
genotype: WT
developmental stage: e13.5
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| Growth protocol |
The brains from e13.5 mouse fetuses were separated at the cervical spinal cord level, and the ganglionic eminences were dissected and harvested. The harvested tissue pieces were collected in complete NSC medium (NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements at a 9:1 ratio; Stemcell Technologies) and dissociated thoroughly but gently by pressing the pipette tip to the bottom of the tube and pipetting five times to obtain a single-cell suspension. The cells were plated at the density of 2x105 cells/ml in complete NSC medium supplemented with 20 ng/ml epidermal growth factor (Stemcell Technologies). Five to six days later, the neurospheres were treated with AccutaseTM (Stemcell Technologies) and cultured for additional 5-6 days. The neurospheres of the second passage were used for experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were crosslinked with 66.7 µl 16% formaldehyde (1% final) at room temperature for 15 minutes. They were quenched with 107 µl 1.25 M glycine (0.125 M final) at room temperature for another 10 minutes in rotating tubes. The samples were centrifuged, and the pellets were washed twice with ice-cold PBS. The crosslinked cells were suspended in 300 µl swelling buffer (50 mM Tris (pH 8.0), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and incubated on ice for 20 minutes to release nuclei. The nuclei were harvested by centrifugation, resuspended in 200 µl ChIP sonication buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 0.5% SDS), and incubated on ice for 20 minutes. The nuclei were sonicated with a probe-based sonicator (FB120, probe:CL-18, FISHER SCIENTIFIC) at a 20% amplitude setting. The sonication was conducted using 15-second pulses at 15-second intervals for a total of 3 minutes. The sonicated nuclei were harvested by centrifugation, and then diluted with 800 µl ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris (pH 8.1), 167 mM NaCl) to reach the final SDS concentration of 0.1%. These samples represented sonicated chromatin ready for immunoprecipitation. To quantify chromatin and assess the degree of its fragmentation, we treated 5% of the sonicated nuclei (50 µl/sample) with 10 µg of RNase A for 15 minutes at 37 ºC followed by 20 µg of proteinase K for 30 minutes at 65 ºC. They were de-crosslinked for 5 minutes at 95 ºC. DNA was extracted with the QIAquick PCR Purification Kit. DNA concentration was determined with a Nanodrop 1000 Fluorospectrometer. DNA fragment sizes were confirmed to be 200-800 bp in length, according to electrophoresis. For immunoprecipitation, an equal amount of sonicated chromatin, based on their prior DNA measurements, of different samples was reacted with anti-RPB1 N-terminal domain Ab (D8L4Y) (1:100) at 4 ºC overnight, followed by 40-µl magnetic protein G beads (Bio-Rad) for another 2 hours at 4 ºC. The beads were rinsed with wash buffer (100 mM Tris (pH 8.0), 500 mM LiCl, 1% NP-40, 1% deoxycholic acid) for 5 times and then with TE buffer once. The chromatin was eluted with elution buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 1% SDS) at 65 ºC for 10 minutes. The immunoprecipitated chromatins were de-crosslinked at 65 ºC overnight with NaCl adjusted to 200 mM. The chromatins were then treated with 10 ug RNase A/sample at 37 ºC for 1 hour, followed by 200 ug proteinase K/sample for 2 hours at 45 ºC. DNA of the samples was purified with QIAquick PCR Purification kit and quantified by the Bioanalyzer (Agilient).
Libraries were prepared robotically with 0.2 to 2 ng of fragmented DNA ranging 100-300 bp in length, using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), as per the manufacturer’s recommendations. Adapters and PCR primers were purchased from Integrated DNA Technologies . Size selection was carried out using SparQ beads (Qiagen) prior to PCR amplification (12 cycles). Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled, and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 225 pM on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2 x 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Each library was sequenced at 25 million reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RPB1(NTD)-specific antibody ChIP DNA fragments
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| Data processing |
Base-calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads. ChIP-seq reads were first trimmed for adapter sequences and low-quality score bases using Trimmomatic (Bolger et al., 2014). The resulting reads were mapped to the mouse reference genome (GRCm38) using BWA-MEM (Li and Durbin, 2009) in paired-end mode at default parameters. Only reads that had a unique alignment (mapping quality > 20) were retained, and PCR duplicates were marked using Picard tools. Peaks were called and annotated using MACS2 (Zhang et al., 2008) and HOMER (Heinz et al., 2010) software suites, respectively.
Genes that lacked RPB1 ChIP-seq signal in the promoter region in the KO tissues were filtered out, as these genes were believed to have no signals in WT tissues neither.
Raw counts were normalized using edgeR’s TMM algorithm (Robinson et al., 2010) and were then transformed to log2 Counts Per Million (log2CPM) using the Voom function implemented in the Limma R package (Ritchie et al., 2015).
To construct the global metagene Pol II-binding profile, normalized read counts (Fragments Per Kilobase of transcript per Million Mapped reads (FPKM) of a full gene length plus 2,000-bp flanks (TSS - 2,000 bp to TES + 2,000 bp) were obtain from all the genes that passed the filtering. Both flanks were divided into 20 equal-sized bins of 100 bp each. The gene bodies were scaled to 60 bins for the full gene length. FPKM was calculated from BAM input files using ngs.plot (Shen et al., 2014) with the following parameters: -G mm10 -R genebody -D ensembl -FL 200 -BOX 0 -SE 1 -VLN 0 -LWD 2 -WD 9. These global metagene Pol II binding profiles were only for visualization of differences in Pol II density, and customarily, and customarily, inferential statistics was not conducted for such profiling.
Genome_build: GRCm38
Supplementary_files_format_and_content: bigwig files contains the profile of ChIP-Seq. NSC_TSS_counts_data.csv, NSC_GENEBODY.counts_data.csv, NSC_TES.counts_data.csv contain the raw counts of RPB1 signal In evry detected gene. NSC_counts_data_tsv contains the raw counts of RPB1 signal in every gene. NSC.log2cpm_data.tsv contains log2CPM value of RPB1 signal in every gene.
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| Submission date |
Mar 24, 2021 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Jiangping Wu |
| E-mail(s) |
jianping.wu@umontreal.ca
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| Organization name |
CR.CHUM
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| Street address |
900 Rue Saint Denis
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| City |
Montreal |
| ZIP/Postal code |
H2X0A9 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE169582 |
RPB1 ChIP-Seq of NSC from Wild Type (WT) and Armc5 knockout mice |
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| Relations |
| BioSample |
SAMN18475743 |
| SRA |
SRX10439067 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5210277_WT_NSC_NTD_2.bw |
688.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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