|Public on Mar 23, 2014
|cell type: Human Skeletal Muscle Myoblasts (HSMM)
hour post serum-switch: 0
control well: FALSE
cells in well: 1
library protocol: Single-cell
|HSMM cells were differentiated for the via switch to 2% HS aMEM (Lifetech).
|Human Skeletal Muscle Myoblasts (HSMM) derived from quadricep biopsy (Lonza, catalog #CC-2580, lot #257130) were expanded in 10% FBS SkGM-2 (Lonza). All procedures have been performed using HSMM within P10 from explant.
|For single-cell mRNA sequencing, dissociated cells were captured and processed with the C1™ Single-Cell Auto Prep System (Fluidigm) following manufacturer’s protocol 100-5950. Starting with a suspension of cells at a concentration of approximately 250 cells/µL, up to 96 single cells are captured in each C1 microfluidic device.
[TruSeq] - HSMM in either growth or differentiation medium were dissociated, washed and resuspended in complete media containing 0.1U/ul of RNaseOUT (Lifetech). Each time point was collected in three independent biological replicates for regular mRNA sequencing while for single cell mRNA sequencing the independent replicates were pooled in equal amounts. For bulk RNA-Seq libraries, total RNA was extracted with Trizol and mRNA libraries were produced starting from 100ng of total RNA using the TruSeq mRNA-Seq library kit (Illumina) according to manufacturer’s instructions.
[Single Cell] - After imaging with a microscope to identify which sites have captured a single cell, processing of the cells occurs within the C1 instrument to perform the steps of cell lysis, cDNA synthesis with reverse transcriptase, and PCR amplification of each cDNA library. The cDNA synthesis and PCR use reagents from the SMARTer® Ultra™ Low RNA Kit for Illumina® Sequencing (Clontech 634936). The SMARTer chemistry utilizes a strand-switching mechanism so that both the 1st and 2nd strands of cDNA are synthesized in a single reaction. Following harvest from the C1 microfluidic device, each cDNA library is subjected to tagmentation (simultaneous fragmentation and tagging with sequencing adapters) using the Nextera XT DNA Sample Preparation Kit (Illumina FC-131-1096) as described in Fluidigm protocol 100-5950. PCR amplification of the tagmented cDNA uses Index Primers from the Nextera XT DNA Sample Preparation Index Kit (Illumina FC-131-1002). Following PCR, the cDNA libraries from individual cells are pooled and purified using AMPure® XP beads (Agencourt Bioscience Corp A63880) as described in Fluidigm protocol 100-5950.
|Illumina HiSeq 2500
|Reads were mapped with TopHat 2.0.9 and Bowtie 2.0.6. TopHat was provided with GENCODE version 17
Mapped reads were analyzed with Cuffdiff 2.2 to generate normalized per-cell expression profiles.
Cells were pseudo-temporally ordered with Monocle
Supplementary_files_format_and_content: FPKM expression matrix; fpkm_matrix.txt for Cell* samples and truseq_fpkm_matrix.txt for *Bulk RNA-Seq samples
|Nov 19, 2013
|Last update date
|May 15, 2019
|Stem Cell and Regenerative Biology
|7 Divinity Ave
|Pseudo-temporal ordering of individual cells reveals regulators of differentiation