Arichard:Notebook/AR151201
Alginate GMD encapsulated nuclei on DropSeq generator
December 1, 2015
Experiment code: AR151201
Motivation
CPT-seq is a combinatorial tagmentation and PCR on long DNA fragments demonstrated by Illumina in 2014 for haplotyping:
http://www.ncbi.nlm.nih.gov/pubmed/25326703
The enabling concept is that tagmentation of double stranded DNA takes place in multiple steps. Tn5 binds rapidly to double stranded DNA, but gel electrophoresis shows that fragments do not appear until after Tn5 is removed from the template by SDS, EDTA, or heat. The intermediate step, between binding and fragmentation, resembles a bead-on-a-string configuration. It is therefore possible to transfer long, double-stranded DNA fragments loaded to saturation with Tn5 transposome complexes from one reaction to another, e.g., from tagmentation to PCR. CPT-seq uses barcoded transposons in a 96-well plate, followed by random mixing and re-distribution into a second 96-well plate with barcoded PCR primers, followed by size-selection and sequencing. This creates a very large number barcode space very few oligos. A low desired collision rate (e.g., 0.1%) is achieved by randomly under-loading the total barcode space (e.g., 1 - 10%). Fragments with identical barcode combinations can be inferred to have originated from the same long fragment.
This experiment is based on two published methods, Drop-Seq and surfactant-free monodisperse alginate GMD generation:
http://www.cell.com/abstract/S0092-8674(15)00549-8 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505195/
First try had no droplets