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| Status |
Public on Jun 30, 2016 |
| Title |
differentiated KD3 mononucleated cell (MNC) single-nucleus RNA-seq 12 |
| Sample type |
SRA |
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| Source name |
immortalized myoblast KD3
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| Organism |
Homo sapiens |
| Characteristics |
stages: mononucleated cell nucleus
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| Treatment protocol |
Upon reaching 80% confluence, we induced differentiation using high glucose DMEM medium supplemented with 2% FBS and ITS supplement (insulin 0.1%, 0.000067% sodium selenite, 0.055% transferrin; Invitrogen). Fresh differentiation medium was changed every 24hrs. Myotubes and MNCs were harvested at 72 hrs after induction of differentiation.
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| Growth protocol |
KD3 cells were grown on dishes coated with collagen in high glucose DMEM(Gibco) supplemented with 20% FBS (Omega Scientific,inc), 1% Pen-Strep(Gibco), and 2% Ultrasor G (Crescent Chemical Co.)
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cell membrane was ruptured with cold cell lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.005% IGEPAL CA-630) for 4-5 mins to release nucleus and the lysis efficiency was checked by Trypan blue (Lonza) staining.
Single cells and nuclei were isolated using the Fluidigm C1 System. We used the largest integrated fluidic circuits (IFCs) of 17-25mm for KD3 cells. Single nuclei C1 runs were completed using the smallest IFC (5-10mm). After capturing, the IFC was loaded with Clontech SMARTer kit lysis, RT, and PCR amplification reagents. Lysis, RT, and PCR amplification was completed overnight and harvested the following day. After harvesting, libraries were constructed using Illumina’s Nextera XT library prep kit per Fluidigm’s protocol. Constructed libraries were multiplexed and purified using AMPure beads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
KD3_MNC_nucleus_B4_R1
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| Data processing |
reads were aligned using STAR v.2.4.2a with parameters ‘--outFilterMismatchNmax 10 --outFilterMismatchNoverReadLmax 0.07 --outFilterMultimapNmax 10’
gene expression was measured using RSEM v.1.2.12
Genes with TPM >= 1.0 in at least 5 nuclei (for lncRNA analysis) or 10 nuclei (for other analyses) were included in the final matrices.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: tab-delimited text files include TPM or count values for each Sample ...
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| Submission date |
Apr 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Weihua Zeng |
| E-mail(s) |
zwhzjh@yahoo.com
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| Organization name |
UCIrvine
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| Department |
Dev & Cell Biology
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| Lab |
Mortazavi lab
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| Street address |
2300E Bio Sci III
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| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697-2300 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE79920 |
Single-nucleus RNA-seq on undifferentiated human KD3 myoblasts and differentiated myotubes and mononucleated cells. |
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| Relations |
| BioSample |
SAMN04613477 |
| SRA |
SRX1681234 |
| 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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