REVERSE TRANSCRIPTION DURING TEMPLATE EMULSIFICATION
Methods to emulsify cells and/or mRNA with reverse transcriptase at a temperature such that the reverse transcriptase begins making cDNA during the emulsification
1 . A library preparation method, the method comprising:
preparing a mixture that includes cells and reagents for reverse transcription;
vortexing the mixture, wherein during the vortexing
the mixture partitions into aqueous droplets that each include zero or one cell,
the cells are lysed to release mRNA into the droplets, and
reverse transcriptase copies the mRNA into cDNAs; and
amplifying the cDNAs into a library of amplicons.
2 . The method of claim 1 , wherein the mixture includes particles and wherein during vortexing the particle template the formation of the droplets, wherein the vortexing is performed using a vortexer or by pipetting to shear the mixture.
3 . The method of claim 2 , wherein the particles comprise gels that include the reagents therein.
4 . The method of claim 2 , wherein the mixture is aqueous and the method includes adding an oil onto the mixture prior to the vortexing.
5 . The method of claim 2 , further comprising, during the vortexing, heating the mixture to a temperature that promotes activity of the reverse transcriptase.
6 . The method of claim 5 , wherein the temperature is between about 40 and 50 degrees C.
7 . The method of claim 2 , wherein the particles are linked to capture oligos that include a 3′ poly-T region.
8 . The method of claim 7 , wherein the particles further include cDNA capture oligos that have 3′ portions that hybridize to cDNA copies of the mRNA, wherein the 3′ portions include gene-specific sequences or hexamers.
9 . The method of claim 2 , wherein the particles are linked to capture oligos that include one or more primer binding sequences cognate to PCR primers that are used in the amplifying step.
10 . The method of claim 1 , wherein the vortexing is performed on a vortexing instrument.
11 . The method of claim 10 , wherein the vortexing instrument vortexes the mixture at a rate between about 200 and 700 rpm.
12 . The method of claim 10 , wherein the vortexing instrument includes a heater that heats the mixture during vortexing.
13 . The method of claim 2 , wherein each of the particles contain some of the reagents for reverse transcription.
14 . The method of claim 2 , wherein each of the particles serves as a template to initiate formation of aqueous monodisperse droplets in oil, in which each droplet comprises one particle.
15 . The method of claim 2 , wherein during the vortexing: the mixture partitions into the aqueous droplets within about 5 to about 50 seconds, and then the cells are lysed within about 30 seconds to about a few minutes, and then the reverse transcriptase begins to copy the mRNA.
16 . A sample preparation method, the method comprising:
preparing, in a sample vessel, an aqueous mixture that includes nucleic acids and polymerase enzymes;
adding an oil to the sample vessel;
shaking the sample vessel to partition the aqueous mixture into droplets surrounded by the oil; and
synthesizing a DNA copy of at least one of the nucleic acids with the polymerase during the shaking.
17 . The method of claim 16 , wherein the nucleic acids are initially in cells and the shaking step forms droplets that contain the cells, the method further comprising lysing the cells within the droplets to release the nucleic acids into the droplets.
18 . The method of claim 16 , wherein the nucleic acids include mRNA and the polymerase enzymes include reverse transcriptase enzymes.
19 . The method of claim 16 , wherein the aqueous mixture includes a plurality of template particles, wherein shaking the sample vessel causes each template particle to serve as a template in the formation of one of the droplets.
20 . The method of claim 19 , wherein the nucleic acids are initially in cells and the shaking step forms droplets wherein each of the droplet contains one template particle and one or zero cells, the method further comprising lysing the cells within the droplets to release the nucleic acids into the droplets.
21 . The method of claim 20 , further comprising, during the shaking step, heating the aqueous mixture to a temperature that promotes reverse transcription.
22 . The method of claim 20 , wherein the template particles are linked to capture oligos linked to the template particles at their 5′ ends, wherein the 3′ ends of the capture oligos include a poly-T sequence.
23 . The method of claim 22 , wherein each of the template particles contain some of the reverse transcriptase enzymes.
24 . The method of claim 16 , further comprising, after the adding step, loading the sample vessel into an instrument that performs the shaking step.
25 . The method of claim 16 , wherein, during the shaking: the droplets form, cells are lysed within the droplets to release the nucleic acids, template particles capture the nucleic acids, and the polymerase enzymes synthesize the DNA copies.
26 . The method of claim 16 , wherein the aqueous mixture includes a plurality of template particles, wherein the method comprises, after the adding step, loading the sample vessel into an instrument that performs the shaking step and wherein shaking the sample vessel causes each template particle to serve as a template in the formation of one of the droplets.
27 . The method of claim 26 , wherein the nucleic acids are initially in cells and the shaking step forms droplets wherein each of the droplet contains one template particle and one or zero cells, the method further comprising lysing the cells within the droplets to release the nucleic acids into the droplets.
28 . The method of claim 16 , wherein the nucleic acids are mRNAs in cells in the aqueous mixture, wherein the droplets contain the cells; and wherein the polymerase enzymes are provided in template particles within the aqueous mixture, wherein the template particles serve as template to cause formation of the droplets during the shaking.
29 . The method of claim 28 , further comprising—after partitioning the aqueous mixture into the droplets—lysing the cells to release the mRNAs into the droplets.
30 . The method of claim 29 , wherein the template particles are bound to capture oligos that capture the mRNAs and prime extension reactions by which the polymerase enzymes copy the mRNAs.