Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset

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Revision as of 22:43, 14 July 2014 by >B1lake (→‎Analysis of Small Nuclei)
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Overview

  • C1 Run Summary:

File:20chips Samplelist.png

  • Primary Mapping/Analysis done by Rui using our pipeline
  • More in depth analysis by Rui

Average cDNA Yields Across Runs

File:20 C1 Runs cDNA Yields 2.png


  • Circled experiments represent the same batch of nuclei. There is little effect of freezing the nuclei in 10%DMSO or running the nuclei on a small chip rather than the medium chip. Global levels of cDNA were reduced with adding a nuclei stain/wash step to the loading protocol using the DNA Seq Cell Wash buffer. Also, addition of PolydIdC did globally increase cDNA levels even for the 0_Nuc conditions.
  • Examination of basic mapping stats shows that the small chip run 19 was highly variable (likely due to capture of small low RNA containing nuclei), while addition of PolydIdC seemed to stabilize mapping rates. Also, even though the cDNA yields were very high for the 0_Nuc with PolydIdC, most of the reads still mapped to ERCC.

Comparison of C1 Run Variations on the Same Nuclei Batch

  • Using Rui's mapped TPM data (unfiltered sample set), performed analysis using Fluidigmn's Singular software (v3.0.3):
    • Identified outliers based on log2 mapping rates and trimmed genes with TPM less than 2 (LOD1)
    • Ran Auto Analysis for top 100 differentially expressed genes
      • Performed pairwise comparison of this dataset:
      • Performed PCA analysis for this dataset:


File:0227 Nuc Pairwise 2.png


File:0227 Nuc PCA 2.png

  • Can see that none of the conditions significantly affected the gene expression profiles (using top 100 differentially expressed genes) of the captured nuclei and these nuclei retained similar PCA distributions across conditions

Analysis of Small Nuclei

  • Experiment 03042014 involved NeuN-low stained nuclei that were small in size and captured using a small chip.
  • cDNA Yields (above) were very low for these single nuclei
    • Hierarchical cluster analysis using Singular Software showed that these small nuclei (pink icon on left side) group together and express interneuron genes:
    • PCA analysis of these small nuclei show them clustering together with other interneurons:

File:Small Nuclei Analysis HC 1.png


File:Small Nuclei Analysis PCA 1.png


  • Therefore, it appears that interneurons show smaller nuclei and lower levels of RNA compared to projection neurons.

Analysis of Glycerol Freezing

  • See Small nuclei analysis above. Included in this are 0320 (fresh) and 0321 (Glycerol Frozen) samples


Analyses of Gene Clusters

Layer1 Analysis: Interneuron Specific Genes

  • Top 27 genes based on Rui's first layer of sample clustering:
    • Addition of gene information and associated references

File:Gene Clustering Analyses Round 1 Top 27.xlsx