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Status |
Public on Dec 09, 2016 |
Title |
BC05_33 |
Sample type |
SRA |
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|
Source name |
BC05_Single-cell RNA-seq
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Organism |
Homo sapiens |
Characteristics |
patient id: BC05 molecular subtype: human epidermal growth factor receptor 2 positive (HER2+) cell type: primary breast cancer
|
Growth protocol |
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.
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Extracted molecule |
total RNA |
Extraction protocol |
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. 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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Description |
Single-cell RNA-seq
|
Data processing |
A modified reference was generated by merging the three array control RNA spike-ins (ThermoFisher) and the human genome reference (hg19) with the GENCODE 19 annotations. Paired-end 100bp reads were aligned to the modified reference by 2-pass mode of STAR_2.4.0b with default parameters. Transcripts Per Million (TPM) values were estimated using RSEM v1.2.17 with default parameters. Genome_build: hg19 Supplementary_files_format_and_content: A processed file is tab-delimited and contains the raw TPM values for 549 single cells and 14 bulk tumors.
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|
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Submission date |
Nov 15, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Woosung Chung |
Organization name |
Samsung Genome Institute
|
Street address |
Samsung Medical Center
|
City |
Seoul |
State/province |
State... |
ZIP/Postal code |
ASI|KR|KS013|SE |
Country |
South Korea |
|
|
Platform ID |
GPL16791 |
Series (1) |
GSE75688 |
Single cell RNA sequencing of primary breast cancer. |
|
Relations |
BioSample |
SAMN06017883 |
SRA |
SRX2350188 |