NextSeq 500 paired end sequencing; GSM2108328: undifferentiated KD3 myoblast single-cell RNA-seq 1; Homo sapiens; RNA-Seq

undifferentiated KD3 myoblast single-cell RNA-seq 1

We report the application of single-nucleus-based sequencing technology for high-throughput profiling of transcriptome in immortazalized human myoblast KD3. By obtaining over sixty billion bases of sequence from mRNA, we generated comprehensive transcriptome profiles from KD3 undifferentiated myoblast and differentiated  multi-nucleated myotube and mono-nucleated cells. We find that the data from single-nucleus RNA-seq is consistent with the transcriptome from single-cell RNA-seq.  The pri-mRNA expression characterized by single-nucleus RNA-seq can reflect the actual miRNA level in the whole cell. Overall design: Examination of transcriptome in 1 cell type in 3 differential stages.

Single-nucleus RNA-seq on undifferentiated human KD3 myoblasts and differentiated myotubes and mononucleated cells.

Submitted by Gene Expression Omnibus on 01-JUL-2016

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.

Single-nucleus RNA-seq on undifferentiated human KD3 myoblasts and differentiated myotubes and mononucleated cells.

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