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SRX997032: GSM1659043: Brx-11_CL#15; Homo sapiens; RNA-Seq
6 ABI_SOLID (AB 5500xl Genetic Analyzer) runs: 15M spots, 751.5M bases, 410Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: A microfluidic device for label-free, physical capture of circulating tumor cell-clusters
show Abstracthide Abstract
Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. Overall design: We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.
Sample: Brx-11_CL#15
SAMN03486740 • SRS911297 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: AB 5500xl Genetic Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters. In addition, two leukocytes were isolated from the blood of a healthy donor using a micromanipulator, after enrichment of the mononuclear fraction using a BD Vacutainer CPT Cell Preparation Tube with Sodium Citrate and staining with the above leukocyte cell surface markers. 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
Experiment attributes:
GEO Accession: GSM1659043
Links:
External link:
Runs: 6 runs, 15M spots, 751.5M bases, 410Mb
Run# of Spots# of BasesSizePublished
SRR19759802,612,332130.6M70.9Mb2015-06-16
SRR19759812,595,001129.8M71.3Mb2015-06-16
SRR19759822,242,990112.1M61.1Mb2015-06-16
SRR19759832,590,422129.5M70.5Mb2015-06-16
SRR19759842,444,186122.2M66.1Mb2015-06-16
SRR19759852,544,671127.2M70Mb2015-06-16

ID:
1447875

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