|
| Status |
Public on Apr 08, 2020 |
| Title |
FLUIDIGMC1_SCATACSEQ_SENS-GFP_CELL_256 |
| Sample type |
SRA |
| |
|
| Source name |
L3 Drosophila eye-antennal disc
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: F2-B(sens)-GFP [w;F2-B/CyO (ato-targeted intronic sens enhancer)]
sequencing method: Single-cell ATAC-seq protocol with the Fluidigm C1 device as described by Buenrostro et al (2015)
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| Growth protocol |
All flies were raised and crossed at 25°C on a yeast based medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Wandering third instar larvae were collected and a total of 200 eye-antennal discs were dissected in ice-cold PBS and placed in SF900 medium. For dissociation, the tissue was placed in 400 µl of trypsin in 0.05% EDTA. The eye–antennal discs were then incubated at 37 °C for 1 h with agitation; being mixed every 20 min with a pipette. After dissociation, cells were centrifuged at 800 g for 5 min at 4 °C and washed with PBS. Finally, the cells were resuspended in 400 µl of PBS, filtered using a 40 µm cell strainer and stained with propidium iodide (PI; final concentration 1 µg/ml) to exclude dead cells. The cells were sorted on a BD Aria I, selecting against the presence of PI and for the presence of GFP.
For single-cell ATAC–seq, as many cells as possible were sorted into a microcentrifuge tube, pelleted by centrifugation at 800 g for 5 min at 4 °C and resuspended at a concentration of 1,000 cells/µl. Single-cell ATAC–seq was performed as previously described12,81, using 5- to 10-µm Open App integrated fluidic circuits (IFCs) on the Fluidigm C1 and with no cell washing step. Briefly, cells were loaded (using a 40:60 ratio of RGT:cells) on a primed Open App IFC (5-10 µm, the protocol for ATAC-seq from the C1 Script Hub was used). After cell loading, the plate was visually checked under a microscope and the number of cells in each of the capture chambers was noted. Next, the sample preparation was performed on the Fluidigm C1 during which the cells underwent lysis and ATAC-seq fragments were prepared. In a 96-well plate, the harvested libraries were amplified in a 25 µl PCR reaction. The PCR products were pooled and purified on a single MinElute PCR purification column for a final library volume of 15 µl. Quality checks were performed using the Bioanalyzer high sensitivity chips. Fragments under 150 bp were removed by bead-cleanup using AMPure XP beads (1.2x bead ratio) (Beckman Coulter).
Single-cell ATAC-seq (Fluidigm C1)
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| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Protocol described by Buenrostro et al (2015)
FLUIDIGMC1_SCATACSEQ_SENS-GFP_CTXREGIONS.tsv
FLUIDIGMC1_SCATACSEQ_SENS-GFP_AGGREGATE.normalized.bw
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| Data processing |
ATAC-seq reads were first cleaned for adapters using fastq-mcf. (ea-utils v1.12) and a list of sequencing primers. Cleaned reads (FastQC v0.1) were then mapped to the 3rd 2017 FlyBase release (D. melanogaster r6.16) genome using Bowtie2 (v2.2.5) with default parameters, and sorted bam files were produced using using SAMtools (v1.2). The data was deduplicated using picard MarkDuplicates.
Aggregation plots were produced using in-house scripts available at: https://github.com/aertslab/ATAC-seq-analysis, and cells well filtered manually based on the aggregation plot profiles, resulting in 72 and sens-GFP+ single cell ATAC-seq profiles. Single-cell profiles were aggregated using samtools merge. Normalized bigwigs were generated using the Kent software (UCSC). Some of the cells were filtered out based on aggregation plots, so they werent use for the analysis (before counting fragments in the regions).
Downstream analysis was done using cisTopic (v0.2.2)
Genome_build: dm6
Supplementary_files_format_and_content: There are two processed data files: a tab-delimited file containing cell names as columns, ctx regions as rows, and number of fragments as values, and a bigwig file containing the normalized coverage profiles of the aggregated sens-GFP+ cells.
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| |
|
| Submission date |
Dec 06, 2019 |
| Last update date |
Apr 08, 2020 |
| Contact name |
Carmen Bravo González-Blas |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB-KU Leuven
|
| Department |
VIB-KU Leuven Center for Brain and Disease Research
|
| Lab |
Laboratory of Computational Biology
|
| Street address |
Herestraat 49
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE141580 |
Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics [FluidigmC1_scATACseq] |
| GSE141590 |
Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics |
|
| Relations |
| BioSample |
SAMN13499531 |
| SRA |
SRX7282542 |
