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| Status |
Public on Sep 07, 2023 |
| Title |
10x scATAC-seq on the mouse liver (0d3236) |
| Sample type |
SRA |
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| Source name |
Liver (P56)
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| Organism |
Mus musculus |
| Characteristics |
tissue: Liver (P56)
genotype: BL/6Jax
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For scATAC-seq we used a modified protocol from the Nuclei Isolation for Single Cell ATAC Sequencing (CG000169) from 10x Genomics. In brief, 200 mg of fresh mouse liver tissue was minced and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (10 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL CA-63, 0.01% Digitonin, 1% BSA) and incubated for 5 min on ice. Next the tissue was homogenized with 15 strokes of pestle A and 15 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). The tissue material was spun down at 500 x g for 5 min and the supernatant was discarded. The tissue pellet was resuspended in 1 mL wash buffer (20 mM NaCl, 20 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 1% BSA). The wash step was repeated one more time and the resulting final pellet was resuspended in 100 µl Diluted Nuclei Buffer (10x Genomics scATAC kit). 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology, and presence of clumps/debris.
Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Single Cell ATAC v1 kit (10x Genomics) according to the manufacturer’s protocol. In brief, the single mouse liver 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: 72°C for 5 min; 98°C for 3 s; 12 cycles of 98°C for 10 s, 59°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA. Next, single-cell droplets were dissolved, and the transposed DNA was isolated using Cleanup Mix containing Silane Dynabeads. 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; 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-seq library was cleaned up with SPRIselect beads (Beckman Coulter). Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on NextSeq500 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.
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Mouse_7_Fresh
Single-cell ATAC-seq (10X Genomics)
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| Data processing |
The generated fastq files were processed with cellranger ATAC (v1.1.0) count function, with include introns=True option. Reads were aligned to Mus musculus reference genome (refdata-cellranger-atac-mm10-1.1.0).
10x scATAC-seq samples were processed with cisTopic,(v0.3.0) using the cells called by cellRanger (5,628 cells) and mm10 SCREEN regions (1,212,823 regions). For topic modelling, we used warpLDA with default parameters, using 500 iterations and inferring models with 2, 5, 10 to 30 (by a step of 1), 35, 40, 45 and 50. This resulted in a model with 19 topics. After correcting sample effects with harmony (v1.0, applied on the scaled topic distributions), we performed Leiden clustering with resolution 0.6, obtaining 11 clusters. The labelled 10x scATAC-seq and multiome cells (annotated based on the transcriptome labels) and the scATAC-seq fragments were used as input for pycisTopic (v1.0.1.dev75+g3d3b721). Briefly, we first created pseudobulks per cell type and performed peak calling using MACS2 (v2.2.7.1, with –format BEDPE –keep-dup all –shift 73 –ext_size 146 as parameters, as recommended for single-cell ATAC-seq data). To derive a set of consensus peaks, we used the iterative overlap peak merging procedure describe in Corces et al. (2018). This resulted in 486,888 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 enrichment > 7, resulting in 22,600 high-quality cells.
Assembly: mm10
Supplementary files format and content: MLV__4aa2e0__Mouse_liver_ctrl_fragments.tsv.gz: Tab-separated text file containing chromosome, start, end, cell barcode and counts for each read for the mouse 6 sample.
Supplementary files format and content: MLV__0d3236__liver_fresh_07_07_2020_fragments.tsv.gz: Tab-separated text file containing chromosome, start, end, cell barcode and counts for each read for the mouse 6 sample.
Supplementary files format and content: scatac_counts.tsv.gz: Tab-separated flat text file containing regions as rows, cells as columns and fragment counts in each region for each cell as values.
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| Submission date |
Nov 21, 2022 |
| Last update date |
Sep 07, 2023 |
| Contact name |
Carmen Bravo Gonzalez-Blas |
| Organization name |
VIB-KU Leuven Center for Brain & Disease Research
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| Street address |
Herestraat 49
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE218467 |
Enhancer grammar of liver cell types and hepatocyte zonation states [scATAC-seq] |
| GSE218472 |
Enhancer grammar of liver cell types and hepatocyte zonation states |
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| Relations |
| BioSample |
SAMN31820818 |
| SRA |
SRX18333072 |
| 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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