Publications

Abstract

The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation.
However, very little is known about its cell type-specific protein composition and the sub-organellar domains where
these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome
proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-
immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts
of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human
iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein
isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry,
processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus
software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of
centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also
be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well.

Abstract

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/β-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.