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Application: RNA-seq analysis
Number of samples: 177
Release date: Apr 12 2016
Last update date: Apr 12 2016
Access: Public
Dataset link Single-muscle-cell transcriptome profiling along the planarian anteroposterior axis

Experimental Protocol


Single-cell RNA-seq on cells isolated from 10 distinct anteroposterior (AP) regions and expressed muscle markers was used to identify muscle regionally expressed genes (mRGs). Non-dividing single cells from 10 consecutive regions along the AP axis were isolated by fluorescence activated cell sorting (FACS), and the resulting single-cell cDNA libraries were screened by qRT-PCR for expression of planarian muscle markers troponin or collagen before sequencing. Principal component analysis (PCA) on the 177 single cells sequenced that expressed >1000 unique transcripts (>2 read count) was performed using highly variable transcripts. Two significant principal components that separated cells by expression of muscle markers (PC1<0) and expression of neoblast markers (PC2<0) were identified. PCA and troponin expression confirmed the identity of 115 muscle cells, and these 115 cells were used in all subsequent analyses. Single-cell differential expression (SCDE, (Kharchenko et al. 2014)) analyses of three different anterior-versus-posterior region comparisons (anterior(regions 1, 2, 3; n=23 cells) versus posterior (regions 8, 9, 10; n=38 cells); head (region 1; 11 cells) versus post-pharyngeal (regions 7, 8, 9; n=35 cells); pre-pharyngeal (regions 2, 3, 4; n=22 cells) versus tail (region 10; n=12 cells)) identified transcripts of 99 genes as differentially expressed at p 2.58). Whole mount in situ hybridization to visualize gene expression patterns verified 44 genes as regionally expressed (mRGs) (35/44 with p<0.005 in any of anterior-versus-posterior SCDE analyses).

Repositories


GEO

GSE74360

ENA

SRP065271

BioProject

PRJNA299816

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