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| Status |
Public on Apr 17, 2023 |
| Title |
10x multiome (ATAC) on the mouse cortex using the 'TST' protocol |
| Sample type |
SRA |
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| Source name |
Cortex (P56)
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| Organism |
Mus musculus |
| Characteristics |
tissue: Cortex (P56)
genotype: 6Jax
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
µl of Protector RNase inhibitor (Sigma)). Nuclei concentration was assessed by the LUNA-FL Dual Fluorescence Cell Counter.
Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and NextGEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer’s protocol. In brief, the single mouse brain nuclei 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 nanolitre-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 37°C for 45 min, 25°C for 30 min and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). Next quenching reagent (Multiome 10x kit) was used to stop the reaction. After quenching, single-cell droplets were dissolved and the transposed DNA and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To fill gaps and generate sufficient mass for library construction, the transposed DNA and cDNA were amplified via PCR: 72°C for 5 min; 98°C for 3 min; 7 cycles of 98°C for 20 s, 63°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. The pre-amplified product was used as input for both ATAC library construction and cDNA amplification for gene expression library construction. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 7-9 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 4°C. The sequencing-ready ATAC library was cleaned up with SPRIselect beads (Beckman Coulter). Barcoded, full-length pre-amplified cDNA was further amplified via PCR: 98°C for 3 min; 6-9 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 5-16 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready GEX library was cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analysed using the Bioanalyzer high-sensitivity chip. All 10x Multiome ATAC libraries were sequenced on NovaSeq6000 instruments (Illumina) with the following sequencing parameters: 50 bp read 1 – 8 bp index 1 (i7) – 16 bp index 2 (i5) - 49 bp read 2. All 10x Multiome GEX libraries were sequenced on NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.
Single-cell Multiome (10X Genomics)
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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 |
The generated fastq files were processed with cellranger-arc (v2.0.0) count function, with include introns =True option. Reads were aligned to Mus musculus reference genome (ata-cellranger-arc-mm10-2020-A-2.0.0).
scRNA-seq data was first analyzed using VSN (0.27.0). Briefly, cells with at least 100 genes expressed and less than 1% of mitochondrial reads were kept. Doublets were removed using Scrublet, with default parameters. 50P PCs were used as input for harmony, which was used to correct batch effects due to the sample preparation protocol, and the corrected PCs were used for dimensionality reduction and leiden clustering (resolution 1). This resulted in 41 clusters, that were annotated based on marker gene expression. Cell types not belonging to the cortex (such as medium spiny neurons from the striatum) were removed, and the data set was reanalyzed using Seurat (4.0.3), using 52 PCs as input for harmony, as previously described, which were used for dimensionality reduction. This resulted in a data set with 21,969 high quality cells (based on scRNA-seq). These cell type labels were used to create pseudobulks from which peaks were called with MACS2 and consensus peaks were derived using the iterative filtering approach (as previously described), resulting in 568,403 regions. We further filtered the data set based on the scATAC-seq quality as well, keeping cells with at least 1,000 fragments, FRiP > 0.4 and TSS > 4, resulting in 19,485 cells.
mm10
Supplementary files format and content: 10x_complex_UC_atac_fragments.tsv.gz: Tab separated file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the '10x complex UC' protocol was used
Supplementary files format and content: 10x_complex_atac_fragments.tsv.gz: Tab separated file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the '10x complex' protocol was used
Supplementary files format and content: 10x_no_perm_atac_fragments.tsv.gz: Tab separated file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the 'no permeabilization' protocol was used
Supplementary files format and content: TST_NP40_004_atac_fragments.tsv.gz: Tab separated file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the 'TST NP40 004' protocol was used
Supplementary files format and content: 10x_TST_atac_fragments.tsv.gz: Tab separated file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the 'TST' protocol was used
Supplementary files format and content: scATACseq_Mouse_cortex_fragment_counts.tsv.gz: tab seperated flat text file containing regions as rows, cells as columns and fragment counts in each region for each cell as values.
Supplementary files format and content: Mouse_cortex_DGEM.tsv: tab seperated flat text file containing genes as rows, cells as columns and counts for each gene in each cell as values.
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| Submission date |
Aug 08, 2022 |
| Last update date |
Apr 17, 2023 |
| Contact name |
Gert Hulselmans |
| E-mail(s) |
[email protected]
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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 |
GPL24247 |
| Series (2) |
| GSE210747 |
SCENIC+: identification of enhancers and gene regulatory networks using single-cell multiomics (Cortex) |
| GSE210749 |
SCENIC+: identification of enhancers and gene regulatory networks using single-cell multiomics |
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| Relations |
| BioSample |
SAMN30195530 |
| SRA |
SRX16982403 |
| 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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