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Sample GSM2364110 Query DataSets for GSM2364110
Status Public on Feb 14, 2019
Title F9-6h
Sample type SRA
Source name mouse B3 cells
Organism Mus musculus
Characteristics tissue: mouse B3 cells
time-point (hours): 6
fluidigm c1 experiment: 3
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.
Growth protocol ERt2-Ikaros inducible B3 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
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).
Submission date Oct 28, 2016
Last update date May 15, 2019
Contact name Camden Jansen
Organization name UCI
Street address 10631 Rockhurst Ave
City Santa Ana
State/province California
ZIP/Postal code 92705
Country USA
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
BioSample SAMN05951400
SRA SRX2281499

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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