Dataset View [GSE52529]

TitlePseudo-temporal ordering of individual cells reveals regulators of differentiation
ArticleRinn JL,Mikkelsen TS,Livak KJ,Lennon NJ,Morse M,Li S,Pokharel P,Grimsby J,Cacchiarelli D,Trapnell C.The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.Nature biotechnology.2014 Apr
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA229164
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP033135
Overall DesginWe selected primary human myoblasts as a model system of cell differentiation to investigate whether ordering cells by progress revealed new regulators of the process. We sequenced RNA-Seq libraries from each of several hundred cells taken over a time-course of serum-induced differentiation.
SummarySingle-cell expression profiling by RNA-Seq promises to exploit cell-to-cell variation in gene expression to reveal regulatory circuitry governing cell differentiation and other biological processes. Here, we describe Monocle, a novel unsupervised algorithm for ordering cells by progress through differentiation that dramatically increases temporal resolution of expression measurements. This reordering unmasks switch-like changes in expression of key regulatory factors, reveals sequentially organized waves of gene regulation, and exposes regulators of cell differentiation. A functional screen confirms that a number of these regulators dramatically alter the efficiency of myoblast differentiation, demonstrating that single-cell expression analysis with Monocle can uncover new regulators even in well-studied systems.
Experimental ProtocolFor 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.
Data processingReads 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; Genome_build: hg19; Supplementary_files_format_and_content: FPKM expression matrix; fpkm_matrix.txt for Cell* samples and truseq_fpkm_matrix.txt for *Bulk RNA-Seq samples
Public OnPublic on Mar 23 2014

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