With Valentines day just behind us, a new genome-wide assay has reinforced the perks of pairing up for single-cells. While multiomic methods have been gaining momentum, it has been tricky for researchers to play matchmaker with ChIP-seq at the single-cell level. To meet this challenge, the lab of Bing Ren at the University of California San Diego developed a new technique they call Paired-Tag (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing).
Previously, the Ren lab has pioneered chromatin conformation capture technologies, adapting some for use in single-cells. In their latest, the group focused on extending a technique they previously developed (Paired-seq), which pairs chromatin accessibility and RNA-seq in single cells. This method works by ligating specific barcodes to open chromatin and cDNA from each cell, followed by sequencing. By adapting the tagmentation and cleavage steps, they have optimized the approach to pair single-cell ChIP-seq and RNA-seq. Here are the details:
- The Paired-Tag protocol can be broken down into 4 key steps:
- Antibodies against a specific histone modification are used to target Tn5 transposase to chromatin
- Tagmentation (cleavage and barcode tagging of chromatin DNA and cDNA by Tn5) and reverse transcription are performed
- A ligation-based combinatorial barcoding strategy introduces second and third rounds of DNA barcodes; nuclei are pooled and separated into wells creating unique barcodes for each nucleus
- The chromatin DNA and cDNA are purified, amplified, and sequenced
To test Paried-Tag in heterogeneous tissue, they used it to integrate H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K9me3 in adult mouse frontal cortex and hippocampus:
- They recovered up to ~20,000 unique loci mapped per nucleus for ChIP-seq and up to ~15,000 unique molecular identifiers per nucleus for RNA-seq
- Nuclei clustered into 22 cell groups based on RNA-seq data; however, clustering based on each histone mark revealed fewer (3-18) clusters, indicating similar profiles for some of these marks in different cell types. Joint histone mark/RNA-seq clustering identified more clusters for most marks
- For each cell group, they combined the ChIP-seq profiles across marks and classified gene promoters into 7 chromatin states (e.g. class I heterochromatin marked by H3K9me3 etc.)
- Finally, they linked chromatin state at cis-regulatory elements to putative target genes expression
Paired-Tag provides joint profiling of the transcriptome and histone marks in single cells for a much lower cost than current methods. Compared to single-cell ATAC-seq, the integration of multiple histone marks in Paried-Seq provides much more information about the state of regulatory elements. The authors are hopeful to pair up multiple histone marks in the same single-cells in the near future by making some tweaks to their protocol.
Catch all the pairings in Nature Methods, February 2021