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Sample GSM8152008 Query DataSets for GSM8152008
Status Public on Mar 21, 2024
Title smart_seq2_3
Sample type SRA
 
Source name K562
Organism Homo sapiens
Characteristics cell line: K562
cell type: chronic myeloid leukemia cells
treatment: none
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.
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.
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
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
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.
 
Submission date Mar 18, 2024
Last update date Mar 21, 2024
Contact name Xi Zeng
E-mail(s) ivociel@outlook.com
Organization name Xiamen University
Street address No. 422, Siming South Road
City Xiamen
ZIP/Postal code 361005
Country China
 
Platform ID GPL24676
Series (1)
GSE261788 AMAR-seq: automated multimodal sequencing of DNA methylation, chromatin accessibility, and RNA expression with single-cell resolution
Relations
BioSample SAMN40517737
SRA SRX23975618

Supplementary file Size Download File type/resource
GSM8152008_smart_seq2_3_counts.txt.gz 188.3 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA

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