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| Status |
Public on Feb 07, 2020 |
| Title |
Reanalysis Brx66_2_SC1_012913 |
| Sample type |
SRA |
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| Source name |
CTCs
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| Organism |
Homo sapiens |
| Characteristics |
patient: Brx66
overall survival days: 1007
overall survival event: 0
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| Treatment protocol |
Patients with a diagnosis of ER and/or PR positive metastatic breast cancer provided informed consent for de-identified blood collection, as per institutional review board approved protocol (DF/HCC 05-300) at Massachusetts General Hospital. Approximately 6-12 mL of fresh whole blood was processed through the microfluidic CTC-iChip as previously described [PMID: 24577360] within 4 hours from the blood draw. Before processing, whole blood samples were incubated with biotinylated antibodies against CD45 (R&D Systems, clone 2D1), CD66b (AbD Serotec, clone 80H3) and followed by incubation with Dynabeads MyOne Streptavidin T1 (Invitrogen) to achieve magnetic labelling of white blood cells. This mixture was processed through the CTC-iChip. To identify CTCs, the CTC-enriched product was stained with Alexa Fluor 488-conjugated antibodies against EpCAM (Cell Signaling Technology, #5198), Cadherin 11 (R&D Systems, FAB17901G), and HER2 (BioLegend, #324410). To identify contaminating white blood cells the product was stained with TexasRed-conjugated antibodies against CD45 (BD Biosciences, BDB562279), CD14 (BD Biosciences, BDB562334), and CD16 (BD Biosciences, BDB562320). The stained product was viewed under a fluorescent microscope where single CTCs were identified based on intact cellular morphology, Alexa Fluor 488-positive staining and lack of TexasRed staining. Cells of interest were individually micromanipulated with a 10 mm transfer tip on an Eppendorf Transfer-Man NK 2 micromanipulator and lysed.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted 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).
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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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Description |
GSM1253390
GSM2314667
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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). See platform.xls for a description of the IDs in readCounts.xls
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| Submission date |
Jan 29, 2020 |
| Last update date |
Feb 07, 2020 |
| Contact name |
Ben S. Wittner |
| E-mail(s) |
wittner.ben@mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital
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| Department |
Center for Cancer Research
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| Lab |
Lawrence
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| Street address |
149 13th Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platform ID |
GPL16288 |
| Series (2) |
| GSE143626 |
Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis |
| GSE144494 |
Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis [rna-Seq CTCs 1] |
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| Relations |
| Reanalysis of |
GSM1253390 |
| Reanalysis of |
GSM2314667 |
| BioSample |
SAMN13941267 |
| SRA |
SRX7648234 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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