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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 28, 2024 |
| Title |
FA4，H3K9me3 |
| Sample type |
SRA |
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| Source name |
sv129
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| Organism |
Mus musculus |
| Characteristics |
cell line: sv129
cell type: Mouse embryonic stem cells (mESCs)
cut&tag antibody: H3K9me3 (CST, 13972)
treatment: normal folic acid(FA=4）
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| Treatment protocol |
1.Establishment of NTD Mouse Model: MTX is a folate antagonist. Under conditions of low folate diet in pregnant mice, we intraperitoneally injected MTX (1.5 mg/kg) at gestational day 7.5. We began observing the closure status of the neural tube in fetal mice during the neural tube closure window at E9.5-10.5. Normal mice appeared plump and round, with fully closed neural tubes, and were categorized into the normal group. Embryos from mice on low folate diet and injected with MTX exhibited varying degrees of developmental delay, smaller embryos, and primarily showed phenotypes of hindbrain malformations and spina bifida, and were categorized into the NTD group.
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| Growth protocol |
Constructing a low-folate cell line. Mouse embryonic stem cells (mESCs) Sv/129 were maintained in Dulbecco’s modifed Eagle’s medium (DMEM, Gibco, USA) supplemented with 0.1 mM β-mercaptoethanol (Invitrogen, Carlsbad, USA), 0.1 mM non-essential amino acids (Invitrogen, Carls_x0002_bad, USA), 0.1 mM glutamate (Invitrogen, Carlsbad, USA), 15% fetal bovine serum (Gibco, USA), and 1000 U/ml mouse leukemia inhibitory factor (Millipore, Bill_x0002_erica, USA), cultured in culture dishes coated with 0.2% gelatin (Invitrogen, Carlsbad, USA). Constructing a low-folate cell line.After resuscitating normal mESCs, Sv129 cells were randomly split into FA4 and FA0. The FA0 group uses folate-free mESCs culture medium.Folate (Sigma) was added to the medium of FA0 as medium of the FA4 group(final concentration: 4 mg/L). Next, two groups were added correspond medium and were cultured at 37°C with 5% CO2 in a humidified incubator and passaged every 2–3 days. The culture medium was changed daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells/tissues crosslinked with 1% formaldehyde for 10 minutes.CUT& Tag was performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903-01) in accordance with the manufacturer’s instructions.
Briefly, cells were gathered and bound to beads coated with concanavalin A, permeabilized by digitonin, and then incubated with H3K9me3 antibodies (CST, 13969).pA-Tn5 transposase was then incubated with the samples. TheDNA was extracted, amplified, and purified following transposon activation and tagmentation to create a library
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CUT&Tag
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| Data processing |
1 Clean Reads Filtering Reads obtained from the sequencing machines included raw reads containing adapters or low quality bases which would affect the following assembly and analysis. Raw reads would be processed to get high quality clean reads according to three stringent filtering standards: i Removing reads containing adapters; ii Removing reads containing more than 10% of unknown nucleotides (N); iii Removing low quality reads containing more than 50% of low quality (Q-value≤20) bases. 2 Reads Alignment BWA[1] (version 0.7.12) using default parameters was used to align the clean reads from each sample against the reference genome, and only the alignments within 2 mismatches were considered in peak scanning. 3 Strand Cross Correlation Strand cross-correlation of tag density providing a quick assessment of IP-seq data set quality and binding characteristics. It is based on the fact that a high quality IP-seq experiment produces significant clustering of enriched DNA sequence tags at locations bound by the protein of interest, and that the sequence tag density accumulates on forward and reverse strands centered around the binding site. SPP R package was used to calculate the correlation between forward and reverse strands, which can reflect whether the chromatin immunoprecipitation effect is optimal. 4 Peak Detection MACS[2] (version: macs14)( -p 1e-3 -m 10,30) is a computational method that was designed to identify read-enriched regions from ChIP-seq data. Dynamic Possion Distribution was used to calculated p-value of the specific region based on the unique mapped reads. The region would be defined as a peak when p-value<1e-5. Then, the distribute of chromosome distribution, peak width, fold enrichment, significant level and peak summit number were displayed. 5 Peak Related Genes Annotation According to the genomic location information and gene annotation information of peak, peak related genes can be confirmed. Besides, the distribution of peak on different genome regions, such as intergenic, introns, downstream2k, upstream2k and exons) was performed.
Assembly: mouse genome (mm10)
Supplementary files format and content: According to the genomic location information and gene annotation information of peak, peak
Supplementary files format and content: related genes can be confirmed. Besides, the distribution of peak on different genome regions, such as intergenic, introns, downstream2k, upstream2k and exons) was performed.
Library strategy: CUT&Tag
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| Submission date |
May 29, 2024 |
| Last update date |
Nov 28, 2024 |
| Contact name |
Xuejia He |
| Organization name |
Capital Institute of Pediatrics
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| Street address |
2 yabao road
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| City |
beijing |
| ZIP/Postal code |
1 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE268545 |
ALKBH1-mediated DNA N6-methyladenine Modification Regulates H3K9me3-dependent Heterochromatin in Neural Tube Development [Cut & Tag] |
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| Relations |
| BioSample |
SAMN41579663 |
| SRA |
SRX24739309 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8294097_4.final.anno.txt.gz |
736.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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