Nucleic Acid Sequence Analysis from Single Cells
Presented herein are methods and compositions for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and/or beads bearing unique barcodes such as unique molecular barcodes (UMI).
1 . A method of preparing a cDNA sequencing library from a plurality of single cell organelles comprising:
a. spatially separating single cell organelles;
b. releasing mRNA, microRNA, small interfering RNA, ribosomal RNA, and/or mitochondrial RNA from each single cell organelle to provide a plurality of individual RNA samples, wherein the RNA in each individual RNA sample is from a single cell organelle;
c. synthesizing a first strand of cDNA from the RNA in each individual RNA sample with a first strand synthesis primer comprising a tag, thereby incorporating the tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to RNA from a single cell organelle, and wherein the tag comprises an organelle-specific identifier sequence and optionally a unique molecular identifier (UMI) sequence;
d. pooling the tagged cDNA samples from the plurality of single cell organelles;
e. amplifying the pooled, tagged cDNA samples to generate pooled, tagged, double-stranded cDNA; and
f. performing a tagmentation reaction on the pooled, tagged double-stranded cDNA samples by contacting the pooled, tagged cDNA with a plurality of transposome complexes to simultaneously cleave each cDNA and incorporate an adapter into each strand of the cDNA, thereby generating a cDNA library from the plurality of single cell organelles.
2 . The method of claim 1 , wherein the first strand synthesis primer is a mixture of oligo dT primer and randomer primers.
3 . The method of claim 1 , wherein the tag comprises a unique molecular identifier (UMI) sequence.
4 . The method of claim 1 , wherein the tag comprises a first-read sequencing adapter sequence.
5 . The method of claim 4 , wherein the plurality of transposome complexes each comprises a transposase complexed with a transposon comprising a second-read sequencing adapter sequence, wherein the second-read sequencing adapter sequence is different from the first-read sequencing adapter sequence, and wherein the cDNA library comprises a plurality of tagged cDNA fragments having the first-read sequencing adapter sequence on a first strand and the second-read sequencing adapter sequence on a second strand.
6 . The method of claim 5 , further comprising amplifying the tagged cDNA fragments of the cDNA library to generate amplified, tagged cDNA fragments, optionally wherein amplifying the tagged cDNA fragments of the cDNA library comprises adding an additional sequence to the 5′ end of the amplification products.
7 . The method of claim 6 , wherein the additional sequence comprises a primer binding sequence for hybridization to a complementary primer binding sequence on a solid support and amplification of the tagged cDNA fragments on the solid support.
8 . The method of claim 7 , further comprising hybridizing the amplified, tagged cDNA fragments to the complementary primer binding sequence on the solid support, and amplifying the amplified, tagged cDNA fragments on the solid support.
9 . The method of claim 8 , further comprising sequencing the amplification products on the solid support.
10 . The method of claim 1 , wherein the tagmentation reaction comprises contacting the double-stranded cDNA with a transposase mixture comprising a Tn5 transposase.
11 . The method of claim 1 , wherein the plurality of transposome complexes consists essentially of transposomes wherein the transposon comprises a second-read sequencing adapter sequence.
12 . The method of claim 1 , further comprising sequencing the tagged cDNA fragments of the cDNA library, optionally wherein sequencing comprises 3′ tag counting or whole transcriptome analysis.
13 . The method of claim 1 , wherein the first strand synthesis primer:
a. comprises a double-stranded portion, optionally wherein the first strand synthesis primer reduces concatenation byproducts compared to a single-stranded first strand synthesis primer;
b. comprises a region capable of forming a hairpin;
c. comprises a region of RNA; and/or
d. is hybridized to a complementary oligonucleotide, thereby forming a double stranded portion.
14 . The method of claim 1 , wherein the first strand synthesis primer is attached to a bead and the synthesizing comprises synthesizing tagged cDNA samples on the bead.
15 . The method of claim 14 , comprising encapsulating each single cell organelle in a droplet with the bead before releasing the mRNA from each single cell organelle, optionally wherein the releasing and synthesizing of each tagged cDNA sample is performed in the droplet or the pooling the tagged cDNA samples comprises pooling the beads with the attached tagged cDNA samples.
16 . The method of claim 15 , wherein the droplet is generated using a droplet actuator.
17 . The method of claim 1 , wherein spatially separating single cell organelles comprises separating the organelles into spatial compartments before releasing mRNA, microRNA, small interfering RNA, ribosomal RNA, and/or mitochondrial RNA from each single cell organelle.
18 . The method of claim 17 , wherein the organelles are separated into a spatial compartment by fluorescence activated cell sorting (FACS) or are separated into a spatial compartment by being immobilized on a solid surface.
19 . The method of claim 18 , wherein the organelles are immobilized on a solid surface by an antibody that specifically binds to the organelle, wherein the antibody is immobilized on a solid surface, optionally wherein the solid surface is a flow cell or a bead.
20 . The method of claim 1 , wherein the organelles are selected from nuclei, mitochondria, and ribosomes.