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| Status |
Public on Oct 28, 2020 |
| Title |
OCI-AML3_single-cell_19 |
| Sample type |
SRA |
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| Source name |
acute myeloid leukemia cell line OCI-AML3
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| Organism |
Homo sapiens |
| Characteristics |
cell line: OCI-AML3
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| Treatment protocol |
N/A
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| Growth protocol |
Kasumi-1 and OCI-AML3 cells were cultured in RPMI medium plus 10% FBS and 1% penicillin/streptomycin in a humidified 5% CO2 atmosphere.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA for cell bulk controls was extracted from Kasumi-1 and OCI-AML3 cell bulk samples using the AllPrep DNA/RNA Kit (Qiagen)
NGS libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) with 20 ng of partially fragmented epi-gSCAR amplicons (Kasumi-1 single cell 1-7), 50ng ng of fragmented epi-gSCAR amplicons (Kasumi-1 single cell 8-27 and OCI-AML3 single cell 1-20) or 50ng ng of fragmented Kasumi-1 and OCI-AML3 bulk gDNA according to the manufacturer's recommendations with 50% reduction of reaction volumes.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
single-cell DNA
assay variant A
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| Data processing |
For the extraction of DNA methylation from epi-gSCAR data, NGS reads were analyzed using a custom bioinformatic pipeline which was automated using Snakemake (version 5.3.0) in Python (version 3.6). After removal of Illumina adapters, overlapping paired-end reads were merged and non-overlapping reads were converted to singletons using BBMerge to obtain single-read information. Next, GAT-Adapter sequences were removed. The resulting pre-processed merged and unmerged reads were filtered for reads containing either 5´ poly(d)T or 3´ poly(d)A tailed HhaI scars separately (motif: GCGAAAAAA or TTTTTTCGC; Hamming distance = 1). Poly(d)T and poly(d)A tails were removed, resulting in reads containing 5´ or 3´ HhaI scars, respectively (5´-scar file and 3´-scar file). Separately, poly(d)T and poly(d)A tails were removed from GAT-Adapter trimmed reads (all-read file). All trimming and filtering steps were performed using BBDuk. Reads were subjected to quality control by FastQC and 5´ or 3´ HhaI scar-containing reads were mapped to the human assembly GRCh37 (hg19) with BWA-MEM separately with soft trimming enabled. Samtools was used to remove secondary and supplementary alignments and alignments with MAPQ smaller than 10. Alignment intervals were generated with the bamtobed command of bedtools and reduced to the outermost three 5´ or 3´ nucleotides, respectively. Scar intervals were filtered for nucleotide-precise overlap with HhaI sites and CpGs in the human genome and assigned as cut HhaI sites. In order to identify uncut (intact) HhaI sites, the all-read file was aligned accordingly, since all reads can potentially contain intact HhaI sites. Next, all HhaI sites of the human genome were expanded by 1 bp on either side as a safety margin and only completely covered intervals were assigned as uncut HhaI sites. Intact sites overlapping with cut HhaI sites (e.g. GCGCG(A)n-3’) were excluded from the output since complete digestion of sites close to DNA ends cannot be guaranteed. Overlap of uncut with cut sites revealed sites of heterozygous methylation. All other uncut and cut HhaI sites were assigned as methylated or unmethylated, respectively. All CpG or HhaI sites of the human genome, which were covered by WGBS datasets of Kasumi-1 or OCI-AML3, respectively, were defined as informative for epi-gSCAR datasets.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: bedgraph files with methylation values for all covered sites for Kasumi-1 single cell 1-27 and OCI-AML3 single cell 1-20
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| Submission date |
Oct 13, 2020 |
| Last update date |
Oct 29, 2020 |
| Contact name |
Heiko Becker |
| Organization name |
University Medical Center
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| Department |
Department of Medicine I
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| Lab |
Becker
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| Street address |
Breisacher Straße 115
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| City |
Freiburg |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE131723 |
Bisulfite-free epigenomics and genomics of single cells analyzed by methylation sensitive restriction (epi-gSCAR) |
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| Relations |
| BioSample |
SAMN16429815 |
| SRA |
SRX9287080 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4830524_epi-gSCAR_OCI-AML3_SC19_meth.bedgraph.gz |
2.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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