Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset: Difference between revisions

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*C1 Run Summary:
*C1 Run Summary:
[[File:20chips_Samplelist.png|400px]]
[[File:1000hNucBA8.jpg|1200px]]
*Primary Mapping/Analysis done by [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2014-7-3#Primary_analysis_on_20_chips_of_hNuc_BA8 Rui] using our pipeline  
*Primary Mapping/Analysis done by [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2014-7-3#Primary_analysis_on_20_chips_of_hNuc_BA8 Rui] using our pipeline  
*More in depth analysis by [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2014-7-4 Rui]
*More in depth analysis by [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2014-7-4 Rui]


==Average cDNA Yields Across Runs==
==Analyses==


[[File:20_C1_Runs_cDNA_Yields_2.png | 500px]]
[[Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset: Protocol Optimizations]]


 
[[Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset: Layer Specific Gene Expression]]
*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 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2014-7-3#Basic_mapping_statistics 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, performed analysis using Fluidigmn's Singular software:
**Identified outliers based on log2 mapping rates and trimmed genes with TPM less than 2 (LOD1)
**Performed pairwise comparison of each run:
 
[[File:0227_Nuc_Pairwise_1.png | 500px]]
 
**Performed PCA analysis on each run:
 
[[File:0227_Nuc_PCA_1.png |500px]]
 
*Can see that none of the conditions significantly affected the gene expression profiles of the captured nuclei and these nuclei retained similar PCA distributions across conditions
 
== next==

Latest revision as of 01:05, 8 November 2014

Overview[edit]

  • C1 Run Summary:

File:1000hNucBA8.jpg

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

Analyses[edit]

Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset: Protocol Optimizations

Blue:RNA-Seq Analyses:C1 hNuc 1000 Dataset: Layer Specific Gene Expression