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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 14, 2019 |
| Title |
E12-18h.2 |
| Sample type |
SRA |
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| Source name |
mouse B3 cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: mouse B3 cells
time-point (hours): 18
fluidigm c1 experiment: 6
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| Treatment protocol |
Briefly, cells were induced with 20mM of 4-hydroxytamoxifen (4OHT), over the course of 24 hours. Prior to performing single-cell experiments, cells were washed twice with cold 1X PBS. An additional dose of 4OHT was administered at ~12h for collection of 24 hour samples.
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| Growth protocol |
ERt2-Ikaros inducible B3 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cells were isolated using the Fluidigm C1 System. Single cell C1 runs were completed using the smallest IFC (5-10 um) based on the estimated size of B3 cells. Briefly, cells were collected for 0,2,6,12,18 and 24 hour time-points at a concentration of 400 cells/μl in a total of 50 μl. To optimize cell capture rates on the C1, buoyancy estimates were optimized prior to each run. Each individual C1 capture site was visually inspected to ensure single-cell capture and cell viability. After visualization, the IFC was loaded with Clontech SMARTer kit lysis, RT, and PCR amplification reagents.
cDNA was normalized across all libraries from 0.1-0.3 ng/μl and libraries were constructed using Illumina’s Nextera XT library prep kit per Fluidigm’s protocol. Constructed libraries were multiplexed and purified using AMPure beads. The final multiplexed single-cell library was analyzed on an Agilent 2100 Bioanalyzer for fragment distribution and quantified using Kapa Biosystem’s universal library quantification kit.
The library was normalized to 2 nM and sequenced as 75bp paired-end dual indexed reads using Illumina’s NextSeq 500 system at a depth of ~1.0-2.0 million reads per library.
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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 |
Single-cell RNA-seq libraries were mapped with Tophat (Trapnell et al., 2009) to the mouse Ensembl gene annotations and mm10 reference genome. Single-cell libraries with a mapping rate less than 50% and less than 450,000 mapped reads were excluded from any downstream analysis, for all subsequent analysis. Cufflinks (Trapnell et al., 2010) version 2.2.1 was used to quantify expression from single-cell libraries using Cuffquant. Gene expression measurements for each single-cell library were merged and normalized into a single data matrix using Cuffnorm.
Genome_build: mm10
Log-transformed FPKMs are available for cells (124) and time-points (0h and 24h) that were used in the development of an integrative computational analysis using self organizing maps (SOMs).
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| Submission date |
Oct 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Camden Jansen |
| E-mail(s) |
csjansen@uci.edu
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| Organization name |
UCI
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| Street address |
10631 Rockhurst Ave
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| City |
Santa Ana |
| State/province |
California |
| ZIP/Postal code |
92705 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE89280 |
Integrative analysis of single-cell ATAC-seq and RNA-seq using Self-Organizing Maps [scRNA-Seq] |
| GSE89285 |
Integrative analysis of single-cell ATAC-seq and RNA-seq using Self-Organizing Maps |
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| Relations |
| BioSample |
SAMN05951623 |
| SRA |
SRX2281696 |
| 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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