| Series | GSE69405 |
| Title | Single-cell RNA sequencing of lung adenocarcinoma patient-derived cells |
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| Year | 2015 |
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| Country | South Korea |
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| Article | Park WY,Joo KM,Kim J,Nam DH,Eum HH,Chung W,Seo YJ,Kim SC,Lee HO,Lee HW,Kim KT.Single-cell mRNA sequencing identifies subclonal heterogeneity in anti-cancer drug responses of lung adenocarcinoma cells.Genome biology.2015 Jun 19 |
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| PMID | 26084335 |
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| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA285793 |
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| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP059035 |
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| Overall Desgin | We performed single-cell RNA sequencing (scRNA-Seq) together with bulk sequencing by applying Smart-Seq protocol (Ramsköld et al., Nat Biotechnol 2012). Enrichment of cancer cells in PDX from primary tumor (LC-PT-45: bulk RNA-Seq, n=1) was identified by histopathological examination and genomic signatures. Tumor cell-enriched PDX cells (LC-PT-45: scRNA-Seq, n=34; bulk RNA-Seq, n=9) were analyzed, and additional batch (LC-Pt-45-Re: scRNA-Seq, n=43; bulk RNA-Seq, n=7) was obtained to check comparable results. H358 human lung cancer cells (scRNA-Seq, n=50; bulk RNA-Seq, n=1) were used as cell line controls. Another lung cancer PDX case (LC-MBT-15: scRNA-Seq, n=49; bulk RNA-Seq, n=7) was prepared to validate our analytical strategy applied in the LC-PT-45 case. |
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| Summary | To address how intratumoral heterogeneity affects anti-cancer drug responses, we profiled transcriptomes of single cancer cells originating from lung adenocarcinoma patient-derived xenograft (PDX) tumors. |
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| Experimental Protocol | In order to isolate single-cells and amplify initial RNA content enough to transcriptome sequencing, we adopted the C1TM Single-Cell Auto Prep System (Fluidigm, CA, USA) with the SMARTer kit (Clontech, CA, USA). Cells were captured on the C1 chip (17-25 μm) and determined as a live single cell by fluorescence microscopic observation. Quantity and quality of amplified cDNAs from individual single cells were checked by Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) and 2100 Bioanalyzer (Agilent Inc., CA, USA). RNAs from bulk cell samples were also amplified using a SMARTer kit with 10 ng of starting material. For WES, gDNAs were prepared using QIAamp® DNA Mini kit (QIAGEN, CA, USA). Exome sequencing was carried using the SureSelect XT Human All Exon V5 kit (Agilent Inc., CA, USA), according to the manufacturer’s standard protocol.; Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instruction, assayed the quantity and quality, pooled, and then sequenced on the HiSeq 2500 (Illumina) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).  Sequencing of the exome library was carried out on the HiSeq 2500 (Illumina, CA, USA) using the 100bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina) at the Samsung Genome Institute (Seoul, Korea).Â
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| Data processing | RNA-Seq reads were aligned to the human genome reference (hg19) together with splice junction information of each sample using the 2-pass mode of STAR_2.4.0d.; Transcripts Per Million (TPM) was quantified by implementing RSEM v1.2.18 in default mode with Genecode v.19 annotation.; Genome_build: hg19; Supplementary_files_format_and_content: Each row of the tab-delimited text file includes ENSEMBL gene ID, corresponding gene name and gene type for samples.
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| Platform | GPL16791 |
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| Public On | Public on Jun 03 2015 |
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Differential expression gene List between two groups.
