Single cell analysis on Melanoma (PMID:27124452)

Paper: Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016
Organism: Homo sapiens
Cell type: CD45+/- cells (Melanoma, T-cells, B-cells, Macrophages, NK cell, CAFs and Endothelial cells)
Sample number: 4,645
Library preparation: Smarter
DATA: GSE72056

  1. 25PE/Bowtie(1.4.1) -n 0 -e 99999999 -l 25 -I 1 -X 2000 -a -m 15 –S/RSEM
  2. Focus on across all cells combined (average log2(TPM)>4.5) or within
    a single tumor (average log2(TPM)>6 in at least one tumor)

  3. exclude from analysis any cells that did not express at least 2,000 of these 5,948 genes
  4. Inference of CNVs from RNA-seq
  5. tSNE followed by density clustering.
  6. Controlling for inter-tumor differences (???)
  7. PCA (PC1 is technical, PC2 is cell cycle, )
  8. CCND3 in high cell cycle, KDM5B non cycle
  9. PC3&6 is location, PC4&5 is MITF
  10. Variability of T-cell exhaustion related genes (reflect treatment response etc)

Proliferation vs Differentiation in CD4 Th2 condition in vivo&vitro (PMID: 27176874)

Paper: Single-cell analysis of CD4+ T-cell differentiation reveals three major cell states and progressive acceleration of proliferation. Genome Biology. (2016)
Organism: Mus Musculus
Cell type: CD4 T-cell in vivo and vitro
Sample number: 287
Library preparation: Smarter/C1, 75PE

  1. mapped to Ensembl38.70 combined with ERCC / GSNAP / HTSeq / TPM / Poor quality cells are detected by mapping on 37 genes on the mitochondrial genome
  2. Continue reading

Combination of Single cell RNA-seq & Electrophysiological recording (PMID: 26689544)

Paper: Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nature biotechnology. (2016)
Organism: Mus Musculus
Cell types: Aspirates from cortical neurons
Sample number: 83
Library preparation: STRT-seq
DATA: GSE70844

  1. Patchs-seq = Pache-clamp stimulus protocol followed by the aspiration of the entire somatic compartment into the recording pipette Continue reading

STRT-seq reveals the diversity of brain cell types (PMID: 25700174)

Paper: Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science. (2015)
Organism: Mus Musculus
Cell types: somatosensory cortex and hippocampal CA1 region
Sample number: 3,005
Library preparation: STRT-seq
DATA: GSE60361

  1. Detail analysis method about STRT is described here
    a) Remove 3′ bases with low quality, b) Extract barcode from 5′ end, c) Discard reads with <25 base after polyA trimming, d) Discard reads with UMI(Phred score<17), e) At most 9 Gs are removed from 5′ end (template switching), f) Discard reads with <6 non-A bases or dinucleotide repaet with <6 other bases Continue reading