Jason Bielas, Fred Hutchinson Cancer Research Center “Deep profiling of complex cell populations using scalable single cell gene expression analysis”. Early adopter of 10X single-cell RNa-seq platform - find out more at the 10X workshop this afternoon. Initially running experiments on PBMCs and finding multiple clusters, the number of these is very dependant on the number of cells profiled. Changing from 4500 to 16K cells increases resolution and they went up to 68K cells. They used a mixed 293T and Jurkat population to validate the single cell separation and found only ~1% doublets. They are now looking to use this system to recover single cell transcriptomes from mixtures, enumerate and classify different immune cells, and identify novel cell types; future work will loot to count tumour infiltration, investigate cSNPs, etc, ther are lots of potential apps.
Paolo Piazza, University of Oxford “Linking epigenetics and gene expression at single cell levels using SMART-ATAC-seq”. This is still a work in progress, but please Tweet only nice things as David is following the meeting from Oxford! SMARTer cDNA synthesis has been around for a while and is used in many single-cell RNA-seq protocols. ATAC-seq uses transposase to capture open chromatin and determine actively transcribed regions of the genome.
Paolo discussed the thought process in developing the SMART-ATAc protool: no cleanup, single-tube, prioritise the ATAC reaction over cDNA synthesis, differential indexing for DNA and RNA. They found the ATAC protocol is very sensitive to experimental conditions, and you need to push the conditions towards smaller fragments. They are using the ERCC spike-in controls and find that these are very important in QC of the data before differential analysis. However it is difficult to predict library quality without proceeding to sequencing - QC may simply need to be done on the sequence data, but reads are very cheap nowadays.
Andrea Kohn, University of Florida “Epitranscriptomic landscape of single neurons: insights in the memory mechanisms”. Switching grears to explore "the dark matter of the genome".