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Status |
Public on Nov 06, 2016 |
Title |
MGH36_P5_A05 |
Sample type |
SRA |
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Source name |
oligodendroglioma single cell
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Organism |
Homo sapiens |
Characteristics |
cell type: oligodendroglioma single cell
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Treatment protocol |
Fresh tumor tissue was minced with a scalpel and enzymatically dissociated using a gentle papain-based brain tumor dissociation kit (Miltenyi Biotec). Large pieces of debris were removed with a 100 micron strainer, and dissociated cells were layered carefully onto a 5mL density gradient (Lympholyte-H, Cedar Lane labs), which was centrifuged at 2,000 rpm for 10 min at room temperature to pellet dead cells and red blood cells. The interface containing live cells was saved and used for staining and flow cytometry. Tumor cells were blocked in 1% bovine serum albumin in Hanks buffered saline solution (BSA / HBSS), and then stained first with CD45-Vioblue direct antibody conjugate (Miltenyi Biotec) for 30 min at 4C. Cells were washed with cold PBS, and then resuspended in 1 mL of BSA / HBSS containing 1uM calcein AM (Life Technologies) and 0.33uM TO-PRO-3 iodide (Life Technologies) to co-stain for 30 min before sorting. Fluorescence-activated cell sorting was performed on FACSAria Fusion Special Order System (Becton Dickinson) using 488nm (calcein AM, 530/30 filter), 640nm (TO-PRO-3, 670/14 filter), and 405nm (Vioblue, 450/50 filter) lasers. Fluorescence-minus-one controls were included with all tumors, as well as heat killed controls in early pilot experiments, which were crucial to ensure proper identification of the TO-PRO-3 positive compartment and ensure sorting of the live cell population. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singlets. Viable cells were identified by staining positive with calcein AM but negative for TO-PRO-3. Single cells were sorted into 96-well plates containing cold buffer TCL buffer (Qiagen) containing 1% beta-mercaptoethanol, snap frozen on dry ice, and then stored at -80C prior to whole transcriptome amplification, library preparation and sequencing.
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Growth protocol |
no culutring (freshly-resected tumor samples)
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Extracted molecule |
total RNA |
Extraction protocol |
RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter) Libraries from isolated single cells were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications.oligo-dT were used to prime reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Paired-end, 38-base reads were mapped to the UCSC hg19 human transcriptome using Bowtie with parameters "-q --phred33-quals -n 1 -e 99999999 -l 25 -I 1 -X 2000 -a -m 15 -S -p 6", which allows alignment of sequences with single base changes such as point mutation in the IDH1 gene. Expression values were calculated from SAM files using RSEM v1.2.3 in paired-end mode using parameters "--estimate-rspd --paired end -sam -p 6", from which TPM values for each gene were extracted. Expression levels were quantified as Ei,j=log2(TPMi,j/10+1), where TPMi,j refers to transcriptper-million for gene i in sample j, as calculated by RSEM41. TPM values are divided by 10 since we estimate the complexity of single cell libraries in the order of 100,000 transcripts and would like to avoid counting each transcript ~10 times, as would be the case with TPM, which may inflate the difference between the expression level of a gene in cells in which the gene is detected and those in which it is not detected. For each cell, we quantified two quality measures: the number of genes for which at least one read was mapped, and the average expression level of a curated list of housekeeping genes. We then conservatively excluded all cells with either fewer than 3,000 detected genes or an average housekeeping expression (E, as defined above) below 2.5. We then distinguished oligodendroglioma from non-malignant cells by inferrence of copy number variations and excluded the non-malignant cells. For the remaining cells we calculated the aggregate expression of each gene as log2(average(TPMi,1...n)+1), and excluded genes with an aggregate expression below 4, leaving a set of 8008 analyzed genes. Genome_build: hg19 Supplementary_files_format_and_content: tab-delimited text file containing the normalized expression levels (E) for 8008 analyzed genes across 2,594 oligodendroglioma cells that passed QC.
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Submission date |
Jul 08, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Itay Tirosh |
E-mail(s) |
Tirosh.itay@gmail.com
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Organization name |
WEIZMANN INSTITUTE OF SCIENCE
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Street address |
Herzl 234
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City |
Rehovot |
State/province |
NA |
ZIP/Postal code |
7610001 |
Country |
Israel |
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Platform ID |
GPL18573 |
Series (1) |
GSE70630 |
Single cell RNA-seq analysis of oligodendroglioma |
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Relations |
BioSample |
SAMN03847662 |
SRA |
SRX1085599 |
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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