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| Status |
Public on Apr 17, 2023 |
| Title |
10x scATAC-seq on mix of MM050, MM099, MM116, MM001, MM011, MM057 and MM087 |
| Sample type |
SRA |
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| Source name |
melanoma cell lines
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| Organism |
Homo sapiens |
| Characteristics |
tissue: melanoma cell lines
cell line: MM050, MM099, MM116, MM001, MM011, MM057 and MM087
treatment: None
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| Treatment protocol |
None
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| Growth protocol |
Cells were cultured in Ham’s F10 nutrient mux (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 100 µg ml−1 penicillin–streptomycin. Cell cultures were kept at 37°C and 5% CO2. The identity of each line has been determined using RNA-seq and ATAC-seq and cell cultures used for experiments providing data to this study were tested for myoplasm contamination and were found to be negative.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were extracted from the mm lines as follows; Cells were washed with phosphate buffered saline (1x PBS; Thermo Fisher Scienific), detached using trypsin (Thermo Fisher Scientific) and centrifuged at 1,000 r.p.m. for 5 min to remove the medium after this protocol CG000169 (10x Genomics) was followed. Briefly, cells were washed with PBS + 0.04% BSA and cell concentration was determined with the LUNA-FL Dual Fluorescence Cell Counter. For each cell line, 500k cells were resuspended in 100 µl nuclei lysis buffer (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20; 0.1% NP40; 0.01% Digitonin and 1% BSA in nuclease-free water). After 5 min incubation on ice, 1 ml chilled wash buffer was added to the lysed cells (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20 and 1% BSA in nuclease-free water). The lysed cell suspension was centrifuged at 500 rcf for 5 min at 4°C and the pellet was resuspended in 1x nuclei buffer.
Single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell ATAC Library & Gel Bead Kit v1-v1.1 and ChIP Kit (10x Genomics). Briefly, single nuclei suspended in 1x nuclei buffer were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanoliter-scale gel-bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 72°C for 5 min; 98°C for 30 s; 12 cycles of 98°C for 10 s, 59°C for 30 s, 72°C for 1 min; and hold at 15°C. After incubation, single-cell droplets were broken, and the single-strand DNA was isolated and cleaned using Cleanup Mix containing Silane Dynabeads. Illumina P7 sequence and a sample index were added to the single-strand DNA during library construction via PCR: 98°C for 45s; 9-13 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 15°C. The sequencing-ready library was cleaned up with SPRIselect beads.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
scATAC-seq runs reads were mapped to GRCh38 and fragments files generated using cellranger-atac count command (v2.0.0) using default parameters. Fragment files for both libraries can be found under the names: CRM__34940b_fragments.tsv.gz and NML__a56c4b_fragments.tsv.gz. A merged fragment count matrix can be found under the name: "mm_lines_fragment_counts.tsv.gz".
Cell barcodes were demultiplexed based on their cell line identity using demuxlet using cell line specific mutations obtained from bulk ATAC-seq data on individual cell lines. For this, Genotypes of the individual cell lines were called using the bcftools mpileup command (v.1.11; options, --max-depth 8000 and --skip-indels) and bcftools call command (v1.11; options, --multiallelic-caller, -variants-only, --skip-variants indels and --output-type b). Only variants which were SNPs and not homozygous across samples were kept. To run demuxlet the scATAC-seq bam files were filtered to only contains reads covering SNPs and having a cell barcode (based on CB tag), reads were piledup using the popscle dsc-pileup command using default parameters and demuxlet was run using the popscle demuxlet command (using the option –field GT).
Based on the cell barcode to cell line annotation pseudobulk consensus peaks were called and a binary count matrix of chromatin accessibility of cells over consensus peaks was generated using pycisTopic.
Only high quality barcodes corresponding to cells were kept by filtering out cells with less than 3.25 and 3.8 log number of unique fragements per cell, FRIP below 0.5 and TSS enrichment below 4 and 5, respectively for data from NML__a56c4b and CRM__34940b using pycisTopic. Doublets based on Scrublet calls with a threshold of 0.25 were removed.
Using the high quality barcodes and the binary accessibility matrix an imputed accessibility matrix, resolving drop outs, was generated using topic modeling. For this Latent Dirichlet allocation with a collapsed Gibbs samples was used and a model of 30 topics using pycisTopic.
Pseudo multi ome data was generated by bootstrapping 5 cells of the same cell line into multiple meta cells containing both imputed chromatin accessibility data and gene expression data( from GSE134432 ) for the cell lines present in both assays. This data is available in the mm_lines_gene_expression_matrix.tsv and mm_lines_region_accessibility_matrix.tsv.gz file together with meta cell to cell line annotations in mm_lines_cell_metadata.tsv.
hg38
Supplementary files format and content: mm_lines_gene_expression_matrix.tsv: tab seperated flat text file containing gene expression counts for the pseudo multi ome data (rows are meta cells and columns are genes).
Supplementary files format and content: mm_lines_region_accessibility_matrix.tsv.gz: tab seperated flat text file, compressed using gzip, containing imputed chromatin accessibility data for the pseudo multi ome data (columns are meta cells and rows are genomic region coordinates).
Supplementary files format and content: mm_lines_cell_metadata.tsv.: tab seperated flat text file containing meta cell to cell line annotations.
Supplementary files format and content: mm_lines_fragment_counts.tsv.gz: tab seperated flat text file, compressed using gzip, containing raw fragment counts (columns are cell barcodes and rows are genomic coordinates).
Supplementary files format and content: CRM__34940b_fragments.tsv.gz: tab seperated flat text file, compressed using gzip, containig Tn5 fragments from scATAC-seq library CRM__34940b (first three columns describe genomic region coordinates (chrom, start, end), column four contains the cell barcode identifier, column five contains the total number of read pairs associated with this fragment).
Supplementary files format and content: CRM__34940b_fragments.tsv.gz: tab seperated flat text file, compressed using gzip, containig Tn5 fragments from scATAC-seq library NML__a56c4b (first three columns describe genomic region coordinates (chrom, start, end), column four contains the cell barcode identifier, column five contains the total number of read pairs associated with this fragment).
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| Submission date |
Aug 08, 2022 |
| Last update date |
Apr 17, 2023 |
| Contact name |
Gert Hulselmans |
| E-mail(s) |
gert.hulselmans@kuleuven.be
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| Organization name |
VIB
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| Department |
Center for Brain and Disease Research
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| Lab |
Laboratory of Computational Biology
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| Street address |
Herestraat 49 PO Box 602
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE210745 |
SCENIC+: identification of enhancers and gene regulatory networks using single-cell multiomics (cell lines) |
| GSE210749 |
SCENIC+: identification of enhancers and gene regulatory networks using single-cell multiomics |
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| Relations |
| BioSample |
SAMN30195481 |
| SRA |
SRX16981308 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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