| CPC C12N 15/1003 (2013.01) [C12N 9/1247 (2013.01); C40B 40/06 (2013.01); C40B 50/06 (2013.01); C40B 50/10 (2013.01); B82Y 5/00 (2013.01)] | 24 Claims |
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1. A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising:
(a) providing a plurality of isolated single nuclei or cells,
wherein the isolated single nuclei or cells comprise DNA molecules and RNA molecules;
(b) distributing the isolated nuclei or cells into a first plurality of compartments,
wherein each compartment comprises a subset of more than one isolated nuclei or cells;
(c) processing each subset of isolated nuclei or cells to generate indexed nuclei or cells comprising indexed RNA nucleic acids and indexed DNA nucleic acids,
wherein the processing comprises adding to the RNA molecules a first compartment specific index sequence by reverse transcription to result in the indexed RNA nucleic acids,
wherein the adding to the RNA molecules comprises:
contacting each subset with a reverse transcriptase and a first primer that anneals to RNA molecules in the isolated nuclei or cells,
wherein the first primer in each compartment comprises the first compartment specific index sequence to generate the indexed nuclei or cells comprising the indexed RNA nucleic acids,
wherein the processing further comprises adding to the DNA molecules a first compartment specific index sequence by transposition to result in the indexed DNA nucleic acids,
wherein the adding to the DNA molecules comprises:
contacting each subset with a transposome complex,
wherein the transposome complex in each compartment comprises the first compartment specific index sequence; and
wherein the first index sequence added to the DNA molecules and the first index sequence added to the RNA molecules in each compartment are either identical or not identical;
(d) combining the indexed nuclei or cells to generate pooled indexed nuclei or cells;
(e) distributing the pooled indexed nuclei or cells into a second plurality of compartments,
wherein each compartment comprises a subset of more than one indexed nuclei or cells;
(f) processing each subset of indexed nuclei or cells to generate dual-indexed nuclei or cells comprising dual-indexed nuclei acids,
wherein the processing comprises adding to the indexed RNA nucleic acids and the indexed DNA nucleic acids a second compartment specific index sequence by ligation of a ligation molecule,
wherein the ligation molecule comprises the second compartment specific index sequence;
(g) combining the dual-indexed nuclei or cells to generate pooled dual-indexed nuclei or cells;
(h) distributing the pooled dual-indexed nuclei or cells into a third plurality of compartments,
wherein each compartment comprises a subset of more than one dual-indexed nuclei or cells;
(i) processing each subset of dual-indexed nuclei or cells to generate triple-indexed nucleic acids,
wherein the processing comprises producing amplified dual-indexed nucleic acids by linear amplification and adding a third compartment specific index to the amplified dual-indexed nucleic acids,
wherein the third compartment specific index is added to the amplified dual-indexed nucleic acids by ligation, primer extension, amplification, or transposition; and
(j) combining the triple-indexed nuclei acids to generate pooled triple-indexed nucleic acids, thereby producing a sequencing library from the plurality of nuclei or cells.
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