Gene Expression
Nebulas
Gene Expression NebulasSummary: We used single-cell RNA-seq to investigate expression stochasticity in rice mesophyll cells from two homogenous genetic background, as well as their F1 hybrid.
Overall Design: Single cells and pool-and-split cDNA libraries were constructed as published previously(Tang et al., 2010). In brief, individual cells are seeded into lysate buffer by mouth pipette, and reverse transcription reacted directly on the whole cell lysate. We then applied exonuclease I (New England Biolabs) to remove free primers. Next, a poly (A) tail was added to the 3’ end of the first-strand cDNAs using terminal deoxynucleotidyl transferase (Invitrogen). Single-cell cDNAs were then amplified by 33 cycles of PCR. The resulting 100–200 ng of amplified cDNAs were used to construct a sequencing library. Final cDNA libraries (200–2,000 ng depending on the amount of input material) were checked for known marker gene OsRBC (Os12g17600) using qPCR, and after passing quality control, libraries were fragmented with a Covaris S2 system. After fragmentation, we constructed cDNA library using the NEBNext Ultra DNA library Prep Kit for Illumina according to the manufacturer’s instructions. Samples were sequenced using Illumina HiSeq 2000 to obtain > 20 million single-end 100-bp reads per sample.
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | In brief, individual cells are seeded into lysate buffer by mouth pipette, and reverse transcription reacted directly on the whole cell lysate. We then applied exonuclease I (New England Biolabs) to remove free primers. Next, a poly (A) tail was added to the 3’ end of the first-strand cDNAs using terminal deoxynucleotidyl transferase (Invitrogen). |
| Library Construction Protocol: | Single cells and pool-and-split cDNA libraries were constructed as published previously(Tang et al., 2010). |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | SINGLE |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |
| 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 |
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