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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 21, 2024 |
| Title |
mESC_9d_RNA_2 |
| Sample type |
SRA |
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| Source name |
E14, 129/Ola
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14, 129/Ola
cell type: mouse embryonic stem cell
treatment: withdrawal of Leukemia inhibitory factor
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| Treatment protocol |
Mouse embryonic stem cells (mESCs; E14, 129/Ola) were grown on 6-cm culture dishes with MEF feeder cells and cultured in a complete medium supplemented with leukemia inhibitory factor (LIF) to prevent differentiation. To induce undirectional differentiation, mESCs were dissociated and replated onto 0.1% gelatin-coated dishes, and cultured without LIF in a feeder-free manner.
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| Growth protocol |
All cultures were maintained at 37ºC in a humidified incubator with 5% CO2. The human chronic myeloid leukemia cell line K562 were cultured in IMDM, supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin-Glutamine.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
AMAR-seq employs a stepwise strategy to lyse cell and nuclear membranes. Then, the RNA fraction undergoes transcriptome library construction on the chip based on the smart-seq2 protocol. For nuclear , cytosines on the GpC sites in the open chromatin regions are methylated with GpC methylation transferase, M.CviPI, to mark the chromatin accessibility. Subsequent bisulphite conversion converts all normal cytosines to uracil, leaving endogenously methylated CpG and artificially labelled methylated GpC unchanged. Since DNA methylation information occurs predominantly at the CpG site and the endogenous GpC methylation level is less than 2%, it is reasonable to use CG methylation to indicate DNA methylation level. The treated GC methylation marks open regions to obtain bilayer molecular information on the nuclear fraction of the cell, culminating in individual cell tri-omics data.
Subsequently, AMAR-seq employs a stepwise strategy to lyse cell and nuclear membranes. Specifically, glycoproteins expressed on the surface of the cell membrane are used to encapsulate the cell in a layer of ConA beads, where the cell is lysed in a hypotonic environment, with the cytoplasm flowing out while the nucleus remains intact. Neighboring electrodes are then energized to drag away droplets of cytoplasmic RNA and leave the nucleus at the hydrophilic site. Then, the RNA fraction undergoes transcriptome library construction on the chip based on the smart-seq2 protocol, including reverse transcription, cDNA amplification, and Tn5 transposase-based library construction. After nuclear membrane lysis, cytosines on the GpC sites in the open chromatin regions are methylated with GpC methylation transferase, M.CviPI, to mark the chromatin accessibility. Subsequent bisulphite conversion converts all normal cytosines to uracil, leaving endogenously methylated CpG and artificially labelled methylated GpC unchanged. Since DNA methylation information occurs predominantly at the CpG site and the endogenous GpC methylation level is less than 2%, it is reasonable to use CG methylation to indicate DNA methylation level. The treated GC methylation marks open regions to obtain bilayer molecular information on the nuclear fraction of the cell, culminating in individual cell tri-omics data.
smart-seq2 and scNOMe-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The original sequencing data acquired includes 150-bp paired-end sequences. Trim Galore version 0.6.7 was used to remove the adaptor prior to alignment. With hg38/mm10, trimmed clean sequences were aligned and de-duplicated using Bismark version v0.15.0, a bisulfite alignment program that invokes bowtie2 version 2.2.9. The alignment was carried out using non-directional library options as a result of the amplification procedure, using the following settings: -fastq --non_directional -bowtie2. The duplicates in aligned BAM file were removed using the script deduplicate_bismark provided by Bismark with the command: deduplicate_bismark -p –bam Aligned_Sample.bam.
As a supplementary script of bismark, bismark_methylation_extractor was used to operate deduplicated Bismark result files and extract the coverage and methylation information of cytosine positions with command: bismark_methylation_extractor -p --ample_memory --cytosine_report –CX Path_to_genome Deduplicated_Aligned_Sample.bam. The coverage2cytosine script from Bismark was used to extract the methylation status of cytosines specifically in GpC and CpG from the generated coverage files. The GCG context contained in the coverage file represents a cytosine position both in CpG and GpC, which is ambiguous. These ambiguous positions were located using OligoMatch and removed from the coverage files.
Assembly: hg38, mm10
Supplementary files format and content: CpG.cov and GpC.cov files containing methylation information for CpG and GpC sites.
Supplementary files format and content: counts.txt file containing gene expression information.
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| Submission date |
Mar 18, 2024 |
| Last update date |
Mar 21, 2024 |
| Contact name |
Xi Zeng |
| E-mail(s) |
ivociel@outlook.com
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| Organization name |
Xiamen University
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| Street address |
No. 422, Siming South Road
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| City |
Xiamen |
| ZIP/Postal code |
361005 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE261788 |
AMAR-seq: automated multimodal sequencing of DNA methylation, chromatin accessibility, and RNA expression with single-cell resolution |
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| Relations |
| BioSample |
SAMN40517715 |
| SRA |
SRX23975608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8152030_mESC_9d_RNA_2_counts.txt.gz |
202.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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