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| Status |
Public on May 23, 2024 |
| Title |
scATAC_mEBs_d21_crispri_rep2_lane_a |
| Sample type |
SRA |
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| Source name |
WD44 (mouse embryonic stem cells)
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| Organism |
Mus musculus |
| Characteristics |
cell line: WD44 (mouse embryonic stem cells)
cell type: Mouse embryoid bodies
genotype: monoclonal CRISPRi (cAS9-KRAB) cell line
treatment: Day 21
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| Growth protocol |
Exponentially growing mESCs are lifted from the plate (aspirate medium, wash with PBS, add 2.5 mL [for 10 cm plate] 0.05% trypsin , incubate 2 minutes at 37C, deactivate trypsin and triturate to a single-cell suspension with 10 mL pre-warmed SL medium). Cells are then counted and spun down (5 min at 300 g). Supernatant is aspirated and cells are resuspended to 2 M/mL in CA medium (medium for EB induction: DMEM, 10% FBS, 1x MEM non-essential amino-acids, 1x Glutamax , 10-5 beta-mercaptoethanol). Cells are counted again, and density adjusted to 1 M/mL with CA medium. 3 mL (3 M cells) are added to 12 mL of CA medium in 10 cm plates (non gelatinized, non adherent). One the next day, plates are gently agitated to promote cell aggregation. Following induction, embryoid bodies (mEBs) are passaged every two days (no daily medium change). mEBs are collected using a serological pipette and transferred to a 50 mL conical tube (typically three plates are pooled). Leftover mEBs on plates are recovered by a CA medium wash and pooled with in the conical tube. mEBs are left to settle (initially up to 15-20 min, faster as the mEBs grow in size). Once mEBs have settled, medium is aspirated from the top, carefully avoiding disturbing the loose pellet. Fresh, pre-warmed, CA medium is then added to 15 mL/plate and mEBs redistributed to plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single-nuclei preparation for scATAC-seq were prepared from day 21 mEB as follows: At day 21 of mEB cultures, mEBs (2X 10cm suspension plates) are collected into a 50 mL conical tube and washed 2X with 1X PBS (w/o Ca, Mg). After consecutive PBS washes, mEBs are treated with 1.5mL of 0.25% trypsin and incubated in 37C bath with gentle agitation (steady concentric swirls of 50mL concial) for 3 minutes. For further dissociation, mEBs are then gently titurated 10X with a p1000 pipette. Again, incubate at 37C for 3 minutes with gentle agitation. After second incubation, mEBs are gently titurated 10X with a p1000 pipette. Inactivate trypsin digestion with CA medium and filter single cell suspension through a 100um filter into new 50mL conical. Count single cell suspension and aim for >>100K total cells. Spin 50mL conical tube at 300g for 5 minutes. After removing supernatant, wash 1X with 1mL of 1X PBS + 0.04% BSA and gently pipette mix 5X. Transfer to a 1.5 mL tube and spin at 300g for 5 min at 4C. Wash again with 1mL of 1X PBS + 0.04% BSA. Again, spin at 300g for 5 min at 4C. Proceed with 10x "Nuclei isolation for Single Cell ATAC Sequencing" protocol.
scATAC-seq libraries were prepared following 10x Genomics protocol in "Chromium Next GEM Single Cell ATAC Reagent Kits v1.1". Read structure: read 1 50 cycles, index 1 8 cycles, read 2 50 cycles, index 2 16 cycles
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics scATAC-seq
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| Data processing |
Fastq files were generated by running cellranger makefastq. The resulting fastq files were then processed to fragment files using 10x Genomics cellranger count (cellranger-atac-cs version 1.2.0, reference = refdata-cellranger-atac-mm10-1.2.0), which were processed through the ArchR pipeline. Arrow files were created with function createArrowFiles (minTSS=4, minFrags=1000). Two different double scores were computed with function addDoubletScores (LSI based: k=10, knnMethod=”LSI”, LSIMethod=1); UMAP based: k=10, LSImet=1, UMAPparam: n_neighbors=40, min_dist=0.4, metric=”euclidiean”). In addition, we used AMULET (from its v1.0-beta version, running function ATACDoubletDetector.py and adding as problematic region a union of the ENCODE excluded list, segmental duplications, simple repeats, repeat masker, and microsatellites from mm10 obtained from UCSC). Nuclei were then filtered for: TSS enrichment >8 and >1995 fragment counts. Following dimensional reduction (addIterativeLSI: useMatrix = "TileMatrix", name = "IterativeLSI", iterations = 2, varFeatures = 100000, dimsToUse = 1:30), and clustering (AddClusters: reducedDims = "IterativeLSI", method = "Seurat", name = "Clusters", resolution = 0.5), doublets were stringently removed by inspecting distribution of fragment counts, doublet scores (ArchR derived), and AMULET doublet scores per clusters. All nuclei from clusters with anomalously high doublet scores across metrics were removed. In addition, individual nuclei with either >17782 fragment counts, LSI doublet score > 0, UMAP doublet score > 0, or AMULET score > 0.3 (thresholds assessed from the distribution of anomalous doublet clusters) were removed. In the end, 31% of nuclei were removed with these filters, leaving 46408 nuclei passing quality filters (20329 nuclei from replicate 1, 26079 nuclei from replicate 2). The resulting filtered nuclei were dimensionally reduced and clustered (same parameters as above), leading to 9 clusters with >200 nuclei. Clusters with highly correlated accessibility over all peaks (averaged over all cells per cluster, R2 on log-transformed accessibility >0.55) and proximal in the low-dimensional projection were merged. The intermediate endoderm cluster (connecting the visceral and parietal clusters) was kept separate to avoid diluting the signal from the two well-delineated clusters.
Assembly: mm10
Supplementary files format and content: scATAC pseudobulk pileup bw files for these clusters were generated using ArchR’s function getGroupBW (normMethod=“ReadsInTSS”, tileSize=50, maxCells=1E5, ceiling=10). scATAC_mEB_d21_pseudobulk_accessibility_per_cluster: column 1: unique peak id; column 2: chromosome; column 3: peak start; column 4: peak end; column 5: ArchR peak score; column 6 to 12: normalized mean peak accessibility per cluster (count per cell per peak is normalized by number of reads in TSS, and averaged over all cells in respective cluster).
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| Submission date |
May 22, 2024 |
| Last update date |
May 24, 2024 |
| Contact name |
Jean-Benoit Lalanne |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Jay Shendure
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE217683 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters [scATAC_mEBs] |
| GSE217690 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters |
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| Relations |
| BioSample |
SAMN41492699 |
| SRA |
SRX24653287 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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