Antibody-guided Chromatin Tagmentation (ACT-seq) CURRENT STATUS: POSTED

ACT-seq is a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. The protocol described here is intended for use with bulk-cell samples. The single-cell iACT-seq protocol is separate.


Introduction
Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. We have developed ACT-seq to improve the ease with which researchers can profile epigenetic marks in small numbers of or single cells. ACT-seq utilizes Tn5 transposase, which is commonly used to map chromatin accessibility and structure. We fused the N terminus of Tn5 transposase to Protein A (PA) to form a novel fusion protein that is guided to an epigenetic mark or chromatin-bound protein of interest by an associated antibody. After washing away unbound complex, the transposition reaction is initiated by addition of an MgCl2-containing buffer, which results in insertion of sequence tags at sites of bound PA-Tnp. The reaction is terminated by incubation with EDTA and proteinase. The labeled fragments are directly amplified using PCR and sequenced using Illumina HiSeq technology. The entire preparation time from cells to library takes only 5 to 6 hours, which is very quick compared to many existing techniques. The standard, native ACT-seq method is suitable for detecting epigenetic marks associated with active or open chromatin, including some transcription factors like Brd4. Formaldehyde crosslinking can be used with ACT-seq (XACT-seq) to enhance signal for many other chromatin-associated proteins like Brg1 and CTCF as well as transcriptionally repressive epigenetic marks such as H3K27me3. Both 3 methods are described in parallel below. We suggest trying native ACT-seq first and using XACT-seq if your antibody does not produce visible bands or smears in the resulting agarose gel compared to a suitable positive control antibody (H3K4me3, H3K27ac, etc.).

Recombinant PA-Tnp Protein
Expression vector is available from addgene under accession number 121137. See attached Appendices document for expression instructions.

Oligonucleotide Sequence Barcodes
Annealed sequence barcodes used in generating the PA-Tnp-antibody complex. See the attached Appendices document for details.

Library Preparation Barcodes
Oligonucleotide index tags to be used when generating the libraries for sequencing. These were designed using the Illumina Adapter Sequences Document, and the sequences can be found in the Appendices document.

Troubleshooting
If no bands or smears are obvious at ~200 to 600 bp relative to a positive control antibody, consider trying the crosslinking XACT-seq steps described above. Other points of optimization include adjusting the amount of complex added to the cells in step 13 (can be increased up to 4-fold if necessary) and adjusting the number of cells in each reaction. Be sure to include a matching negative control such as 7 IgG for each condition to measure the background signal.
In our hands, recombinant PA-Tn5 loses activity over time when stored at 20 degrees C or warmer. If poor results are obtained using an aliquot that has been stored under these conditions for an extended time, it would be best to try again using a freshly thawed aliquot from the -80 degree C stock.

Supplementary Files
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