Mouse cortical cell taxonomy by single cell RAN-seq (PMID: 26727548)

Paper: Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nature Neuroscience. (2016)
Organism: Mus Musculus
Cell types: Cortical cell
Sample number: 1,809
Library preparation: Smart-seq
DATA: GSE71585

  1. The Allen Brain Atlas data portal
  2. 100SE, RSEM, align unmapped reads to ERCC and tdT sequence using Bowtie
  3. QC: i)Correlation-ERCC RPKM vs molecule counts, ii)mean and SD for mapping ratio to transcriptome, discard mapping ratio 30% of cells isolated at the same time fall QC
  4. use genes with more variance than technical noise (ERCCs)
  5. Clustering is performed with 2 methods
  6. PCA with RPKM, Select relevant PCs whose eigenvalue is greater than the mean 2SD
  7. Cluster cells by Euclidean distance by PCs with Ward’s method, confirm the significance by sigClust in R
  8. Performe above with different technical noise thresholds and select best on PC spectrum and sigClust P value
  9. WGCNA(use TPM), identify genes with more variation than technical noise, WGCNA with power=4 and min cluster siza=10
  10. For each WGCNA gene module, cluster cells based on member genes. Discard modules if the one cluster contains fewer than 4
  11. Detect differentially expressed genes between clusters and discard modules with less DE genes
  12. Clustering with all remaining modules with Ward’s
  13. Sharpen the boundaries of the groups by DE genes among all with limma@R
  14. Identified clusters are validated by machine learning-based cross-validation approach
  15. Exon usage is evaluated by limma and MISO
  16. Validation of RNA expression is done by RNA double fluorescence in situ hybridization and Fluidigm BioMark
  17. Script for analysis above is available
  18. 49 transcriptomic cell types are identified, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal. Validation is done with Cre lines(gene-Cre x Ail4, which express tdTomato after Cre-mediated recombination)
  19. Some of cell types identified show specific electrophysiological and axon projection properties
Posted in Reference, Single cell and tagged .

Leave a Reply

Your email address will not be published. Required fields are marked *