The Tn5 Transposase Takes Single-cell ChIP-Seq to the Next Level

It’s never good to see someone lagging behind the rest, but while stepwise developments to single-cell chromatin immunoprecipitation and subsequent parallel sequencing techniques (ChIP-seq) have led to gradually improved yields and increased sensitivity with reduced numbers of cells since the first report of a single-cell ChIP protocol, we still lack widely-applicable and low-cost methods to sensitively assess chromatin states in vanishingly small numbers of cells.

However, a busy team of researchers led by Aibin He (Peking University, China) have caught up to the other single-cell technologies and have taken single-cell ChIP-Seq to the next level in not one, but two fascinating articles that describe exquisitely-sensitive techniques (single-cell itChIP-seq and CoBATCH) that leverage the power of tagmentation with the Tn5 transposase!

The Tn5 transposase, a member of the RNase superfamily of proteins that includes retroviral integrases found in certain bacteria, has already found use in ChIPmentation (ChIP-Seq via tagmentation) where it acts to fragment the chromatin and simultaneously add primers for barcoding and PCR amplification and unique molecular identifiers to index each read to a  specific cell. Now, the He lab has employed Tn5 tagmentation in the development of two similar and yet contrasting techniques that put the analysis of chromatin states in the smallest of cell samples within reach of most modestly equipped laboratories.

Single-cell itChIP-Seq – Combining Simultaneous Indexing and Tagmentation For Epigenetic Analyses

The first of these new techniques, indexing and tagmentation-based ChIP-sequencing (or itChIP-seq for short), enables the assessment of histone modifications and non-histone protein profiles in small amounts of cells, even down to the single-cell level. Of note, itChIP-seq does not require the specialized microfluidic devices employed in other related techniques, and the development of the single-cell version of the protocol focused on making the entire process affordable and hence widely applicable.

So how does this new technique take single-cell ChIP-Seq to the next level?

• The base itChIP protocol takes place in a single tube to avoid sample loss
  • Fixed and quenched cell samples undergo optimized chromatin opening to avoid the disruption of protein binding to DNA
  • Tn5 then fragments chromatin while simultaneously introducing simple barcoded adapters for PCR amplification and sequencing within intact cells
  • An optimized sonication step then lyses cells to gently release high-yield evenly fragmented barcoded chromatin
  • Finally, the soluble chromatin is ChIP’ed with a specific antibody allowing sequencing of the resulting precipitated DNA
• The “Low Input” version of itChIP functions well for the analysis of permissive histone marks (H3K4me3), repressive histone marks (H3K27me3), and non-histone DNA-binding proteins (p300, RNA Polymerase II, and EZH2) in around 500 mouse embryonic stem cells (ESCs)
  • Comparative analysis as a function of the number of input cells found higher accuracy for itChIP-seq when compared to other similar methods
• “Single-cell” itChIP-Seq employs a slightly more complicated strategy based on combinatorial dual-indexing approaches for single-cell genomic sequencing in a 96-well plate format
  • The Tn5 complex introduces combinations of barcoded adaptors following chromatin opening; chromatin immunoprecipitation and library amplification from indexed cells then creates a second level of barcodes amenable for next-generation Illumina sequencing
  • As an example of the power of single-cell itChIP-Seq, profiling of H3K27ac yields ~9,000 unique reads per cell, with comparisons suggesting higher sensitivity when compared to similar approaches
• The authors then employed single-cell itChIP-Seq of H3K27ac to explore the early epigenetic priming events occurring during cell fate transitions as naïve mouse ESCs convert into to the primed pluripotent state of epiblast-like stem cells
• Finally, with affordability and applicability in mind, the authors then modified the library preparation to allow for the construction of libraries amenable to more standard Illumina sequencing workflows
  • The addition of Truseq PCR handles made the validated sc-itChIP-seq strategy 15-fold cheaper

Overall, the authors believe that their new technique provides a means to understand concepts such as the epigenetic basis of cell-to-cell variability in heterogeneous cell types and stem/progenitor cell fate transitions in development and disease. While they do note the requirement for further innovation to improve sensitivity, the team believe that this report lays the groundwork for widespread application of single-cell ChIP-seq to multiple fields in any laboratory setup.

CoBATCH – Combinatorial Barcoding and Targeted Chromatin Release for High-Throughput Single-Cell Analyses

In the second study and the second new technique from the He laboratory, Wang et al. report on the development and application of combinatorial barcoding and targeted chromatin release (or CoBATCH for short) as an immunoprecipitation-free single-cell ChIP-seq technique for the efficient high-throughput analyses of histone modifications or chromatin-binding proteins. The emphasis here is on “high-throughput,” with the technique designed to capture the histone modification profiles of a few thousand single-cells with starting materials of at least 10,000 cells.

So how does CoBATCH take single-cell ChIP-Seq to the next level?

• The protocol begins with “in situ ChIP”
  • An antibody of choice added to permeabilized cells binds to specific chromatin regions
  • The addition of Protein A fused with Tn5 transposase (PAT) to these cells targets tagmentation activity to antibody binding regions through an interaction between the antibody and the Protein A of PAT
• The activation of Tn5 activity in PAT-tethered chromatin triggers targeted barcoding and chromatin fragmentation for subsequent sequencing library preparation in one tube without the need for DNA extraction and purification
  • This enables low input epigenomic profiling in intact cells, tissues, and organs under native and cross-linked conditions in less than 24 hours
• The development of the high-throughput CoBATCH technique adapts in situ ChIP to 96-well plates (with a 384-well plate version also possible) and again uses a combinatorial dual-indexing approach
  • The addition of a specific antibody to 200 to 2,000 permeabilized cells per well is followed by the addition of PAT for chromatin fragmentation and indexing using a combination of barcodes
  • Cells are then collected, and 20-25 cells are redistributed into each well of multiple additional plates and used for library preparation and sequencing
  • Each secondary 96-well plates can provide epigenetic profiles of around 2,400 single cells
  • CoBATCH delivers up to 12,000 unique non-duplicated reads per cell with extremely low background, and initial results provide similar profiles to those created using standard bulk ChIP-seq
• The authors then applied their technique to examine endothelial cell heterogeneity
  • CoBATCH for H3K27ac to profile 3,000 single endothelial cells from mouse organs at embryonic day 16.5 uncovered heterogeneity, developmental diversification, and cis-regulatory dynamics
  • Analysis of H3K36me3 and RNA polymerase II from cardiac endothelial cells revealed profound cell-to-cell diversity in endothelial cells subtypes and derivatives

Overall CoBATCH permits the high-throughput analysis of thousands of single cells without the requirement for expensive specialized devices; however, the authors do note that this technique does not suit the analysis of fewer than around 10,000 cells – lucky there is another technique for that!

Single-cell ChIP-Seq: Future Developments

The He lab has now taken a few great strides forward in their effort to stop the single-cell ChIP-seq field from lagging behind the rest and have taken it to the next level; but what does the future hold? Authors from both studies describe how they hope to improve both sets of techniques in their highly detailed studies; these include new innovations to improve sensitivity for itChIP and the modification of CoBATCH to allow analyses of smaller cell numbers.

 For yet more information on single-cell itChIP-Seq, head over to Nature Cell Biology, September 2019, and for more on high-throughput analyses with CoBATCH, see Molecular Cell, August 2019.