Gene Expression
Nebulas
Gene Expression NebulasSummary: We performed single cell RNA sequencing (RNA-seq) for 549 primary breast cancer cells and lymph node metastases from 11 patients with distinct molecular subtypes (BC01-BC02; estrogen receptor positive (ER+); BC03; double positive (ER+ and HER2+); BC03LN; lymph node metastasis of BC03; BC04-BC06; human epidermal growth factor receptor 2 positive (HER2+); BC07-BC11; triple-negative breast cancer (TNBC); BC07LN; lymph node metastasis of BC07) and matched bulk tumors. We separated these single cells into epithelial tumor and tumor-infiltrating immune cells using inferred CNVs from RNA-seq. The refined single cell profiles for the tumor and immune cells provide key expression signatures of breast cancer and the surrounding microenvironment.
Overall Design: All single-cell mRNA expression profiles were acquired from eleven patients (BC01-BC11) including two lymph node metastases (BC03LN; BC07LN) (549 samples). We applied four filtering criteria so as to remove samples with low sequencing quality and finally obtained 515 single cell sequencing data. Matched bulk tumor tissues and/or pooled cells were also sequenced and analyzed by the single cell RNA-seq pipeline (14 samples). Bulk tumor transcriptomes showed significant correlations with the average of single cell transcriptomes.
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| Growth Protocol: | On the day of surgery; all single cell were dissociated with mechanical and enzymatical method then immediately used for assay without any in vitro culture.; On the day of surgery; all single cells were dissociated with mechanical and enzymatical method then immediately used for assay without any in vitro culture. |
| Treatment Protocol: | - |
| Extract Protocol: | Bulk RNAs were extracted from ~1x10^5 cell of suspensions or tumor tissues using RNeasy Plus Micro kit (Qiagen) and 10ng of total RNAs were amplified with SMARTer kit (Clonetech) under the same conditions as single cell. All of amplified cDNAs were quantified and qualified by the Qubit? 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies). Total 549 single cell cDNAs and 14 bulk samples were subjected to the RNA sequencing.; Dead cells in single cell suspensions were removed by Ficoll-Paque TM PLUS (GE healthcare) separation before loading to integrated fluidic circuit (IFC) chip. Each suspension was loaded to 10-17um IFC for mRNA sequencing chip then cDNA synthesis and amplification were performed with SMARTer Ultra Low RNA Kit (Clontech) in the C1TM Single-Cell Auto Prep System (Fluidigm). RNA spike-ins 1, 4 and 7 from ArrayControl TM RNA Spikes (ThermoFisher) were added to the lysis mix for validation of array and batch effect. Bulk RNAs were extracted from ~1x10^5 cells of suspensions or tumor tissues using RNeasy Plus Micro kit (Qiagen) and 10ng of total RNAs were amplified with SMARTer kit (Clonetech) under the same conditions as single cells. All of amplified cDNAs were quantified and qualified by the Qubit 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies). Total 549 single cell cDNAs and 14 bulk samples were subjected to the RNA sequencing. |
| Library Construction Protocol: | Starting with 0.375 ng of amplified cDNAs, sequencing libraries were constructed with the Nextera XT DNA Sample Prep Kit (Illumina). All of constructed libraries were quantified and qualified by the Qubit 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies) then pooled without index overlapping. Finally pooled libraries were sequenced using the HiSeq2500 (Illumina) as 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit.; Starting with 0.375 ng of amplified cDNAs, sequencing libraries were constructed with the Nextera XT DNA Sample Prep Kit (Illumina). All of constructed libraries were quantified and qualified by the Qubit 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies) then pooled without index overlapping. Finally pooled libraries were sequenced using the HiSeq2500 (Illumina) as 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| 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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