Systems and methods for epigenetic sequencing
The present invention generally relates to microfluidics and/or epigenetic sequencing. In one set of embodiments, cells contained within a plurality of microfluidic droplets are lysed and the DNA (e.g., from nucleosomes) within the droplets are labeled, e.g., with adapters containing an identification sequence. The adapters may also contain other sequences, e.g., restriction sites, primer sites, etc., to assist with later analysis. After labeling with adapters, the DNA from the different cells may be combined and analyzed, e.g., to determine epigenetic information about the cells. For example, the DNA may be separated on the basis of certain modifications (e.g., methylation), and the DNA from the separated nucleosomes may be sequenced using techniques such as chromatin immunoprecipitation (“ChIP”). In some cases, the DNA sequences may also be aligned with genomes, e.g., to determine which portions of the genome were epigenetically modified, e.g., via methylation.
1. A method comprising:
providing a plurality of cells individually compartmentalized in microfluidic droplets, wherein an individually compartmentalized cell is contained in a first microfluidic droplet, an adapter contained in a second microfluidic droplet,
coalescing the first and the second microfluidic droplet, wherein at least some of the nucleic acid sequences originating from an individually compartmentalized cell become attached to an adapter, the adapter comprising an identification sequence, wherein at least some of the nucleic acid sequences originating from the same cell contain identical identification sequences, and at least some of the nucleic acid sequences originating from different cells contain different identification sequences; and
amplifying at least some of the nucleic acid sequences.
2. The method of claim 1 , wherein the adapter comprises a restriction site.
3. The method of claim 1 , comprising cleaving the adapter at the restriction site.
4. The method of claim 1 , wherein the adapter comprises a primer site.
5. The method of claim 4 , wherein the primer site comprises a poly-T primer site or a universal primer site or both.
6. The method of claim 1 , wherein the primer site comprises a random primer site.
7. The method of claim 1 , wherein the primer site comprises a PCR primer site.
8. The method of claim 1 , wherein the identification sequences comprise 8 nucleotides.
9. The method of claim 1 , wherein a plurality of adapters are used, comprising a plurality of different identification sequences each having the same length, and a substantially identical restriction site.
10. The method of claim 1 , wherein at least some of the cells are prepared from a tissue.
11. The method of claim 1 , wherein the adapter is palindromic.
12. The method of claim 1 , wherein the plurality of nucleic acid sequences comprise RNA which RNA molecules originate from an individually compartmentalized cell are barcoded or ligated with an identification sequence using the adapter.
13. The method of claim 12 , wherein the adapter is coupled to the RNA molecules by direct ligation or by poly-T or random primer-based reverse transcription.
14. The method of claim 1 , wherein the adapter is present on a solid support.