| Series | GSE60407 |
| Title | Human Pancreatic CTCs Express the ECM Protein SPARC |
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| Year | 2014 |
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| Country | USA |
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| Article | Haber DA,Maheswaran S,Toner M,Shioda T,Ramaswamy S,Kapur R,Ferrone CR,Deshpande V,Rivera MN,Bardeesy N,Qu N,Ellis HL,Shahid M,Arora KS,Trautwein J,MacKenzie OC,Zhu H,Ciciliano JC,Xega K,Brannigan BW,Bersani F,Aceto N,Miyamoto DT,Shah AM,Vincent Jordan N,Ligorio M,Wittner BS,Ting DT.Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.Cell reports.2014 Sep 25 |
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| PMID | 25242334 |
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| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA258196 |
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| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP045493 |
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| Overall Desgin | To achieve a deep RNA sequencing profile of CTCs at the single cell level, we applied a novel inertial focusing-enhanced device, the CTC-iChip, which allows high efficiency negative depletion of normal blood cells, leaving unattached CTCs in solution where they can be selected and analyzed as single cells (Pubmed ID 23552373). CTCs were then subjected to single cell RNA-sequencing (Pubmed ID 20203668). |
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| Summary | Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origin. A total of 13 of 60 extracellular protein genes enriched in mouse CTCs (see GEO GSE51372) were expressed at high levels (>100 rpm) in at least one human pancreatic CTC. Human SPARC was the only gene found at high levels in all human pancreatic CTCs. |
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| Experimental Protocol | Blood specimens from 3 patients with PDAC were subjected to microfluidic depletion of RBCs and CD45 and CD66b-positive WBCs, leaving single CTCs and small CTC-clusters in the final product (pubmed ID 23552373). The product containing enriched cells was collected in a 35mm petri dish and viewed using a Nikon Eclipse Ti inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer and immediately flash frozen in liquid nitrogen.; Libraries were constructed as previously described (Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V.I., Lao, K., and Surani, M.A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516-535). Briefly, to generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07uL T4 gene 32 protein, and 0.33uL SuperScript III Reverse Transcriptase per 1X volume) and incubated on thermocycler at 50C for 30 minutes and 70C for 15 minutes. To remove free primer, 1.0uL of EXOSAP mix was added to each sample, which was incubated at 37C for 30 minutes and inactivated at 80C for 25 minutes. Next, a 3'-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6uL 10X PCR Buffer II, 0.36uL 25mM MgCl2, 0.18uL 100mM dATP, 0.3uL Terminal Transferase, 0.3uL RNase H, and 4.26uL H2O per 1X volume) at 37C for 15 minutes and inactivated at 70C for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2uL 10X High Fidelity PCR Buffer, 1.76uL 2.5mM each dNTP, 0.066uL UP2 Primer at 100uM, 0.88uL 50mM MgSO4, 0.44uL Platinum Taq DNA Polymerase, and 13.654uL H2O per 1X volume) at 95C for 3 minutes, 50C for 2 minutes, and 72C for 10 minutes. DNA was sheared using a Covaris S2 system and then prepared for ABI 5500XL library construction with end polishing, size selection of 200-500 bp using AMPure XP, ABI barcode adaptor ligation, amplification and purification with AMPure XP, and then pooling of barcoded samples for emulsion PCR wiht template beads preparation. Samples were then loaded per protocol on the ABI 5500XL.; RNA-Seq using oligo-dT cDNA synthesis and amplification of cDNA libraries using custom universal PCR primers
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| Data processing | Color space reads were aligned using tophat version 2.0.4 and bowtie1 version 0.12.7 with the no-novel-juncs argument set with human genome version hg19 and transcriptome defined by the hg19 knownGene table from genome.ucsc.edu.; Reads that did not align or aligned to multiple locations in the genome were discarded.; The hg19 table knownToLocusLink from genome.ucsc.edu was used to map, if possible, each aligned read to the gene who's exons the read had aligned to. The reads count for each gene was the number of reads that were so mapped to that gene.; The read count was divided by the total number of reads that were mapped to any gene and multiplied by one million to form the reads-per-million (rpm) count.; Genome_build: hg19; Supplementary_files_format_and_content: The readCounts.xls file gives the read counts described above for each sample (columns) and gene (rows). The file platform.xls gives the genes corresponding to the row IDs in readCounts.xls.
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| Platform | GPL16288 |
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| Public On | Public on Sep 22 2014 |
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