Gene Expression
Nebulas
Gene Expression NebulasSummary: Transcriptomic analysis of dedifferentiation in Dictyostelium discoideum by single cell RNAseq
Overall Design: scRNAseq of cells during the first 6 h of dedifferentiation in liquid growth media. Dedifferentiation cultures with staggered start times were harvested simultaneously at 0, 1, 2, 3, 4, 5 and 6 h of dedifferentiation and pooled together for sequencing using the 10x Chromium platform. Cells from different timepoints were pooled before barcoding. Two biological replicates were collected. In each biological replicate, the input cells were split between two Chromium 10x chip inlets (technical replicates) with data from each technical replicate pooled after mapping for downstream analysis. In biological replicate 1, 967 libraries passed UMI filtering by CellRanger v2.2.0 with median of around 25000 total molecular counts. In biological replicate 2, 2961 libraries passed filtering,with a median of around 18000 total molecular counts.
| Strategy: |
|
| Species: |
|
| Healthy Condition: |
|
| Cell Line: |
|
| Development Stage: |
|
| Growth Protocol: | Before development, cells were grown in HL5 (Formedium, UK). |
| Treatment Protocol: | Cells were developed for 14 h on Whatman #50 filter paper in a humified chamber. Developed cells were disaggregated in KK2 + 20 mM EDTA then dedifferentiated in HL5 growth medium as a shaken suspension. Start times of development and dedifferentiation were staggered to allow simultaneous collection of samples into ice-cold KK2 at 0, 1, 2, 3, 4, 5 and 6 h of dedifferentiation. |
| Extract Protocol: | Cell suspensions from different timepoints were pooled before loading onto the 10X Chromium Single Cell A Chip using the Chromium 3' Library and Gel Bead Kit v2. Samples were split between two chip inlets and treated as technical replicates |
| Library Construction Protocol: | The purified GEM-RT product was subjected to 14 cycles of cDNA amplification. 35 microlitres of cDNA was used to prepare the 10X 3' RNA library, with 12/11 cycles used for sample index PCR. Final libraries were run on a NextSeq500 mid-output 150-cycle kit with a 26[8]98 cycle configuration to generate 130 million read pairs in total. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | NextSeq 500 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
|---|