ICO, or ICO-seq is the first adaptation of high-throughput RNA sequencing to microbes. This method assessed microbial function at a genome-wide scale. For engineered microbes, this method can elucidate how changes to the microbial genome can perturb its functions.
Obtain yeast from a suspension culture, and count the cells using a hemocytometer. Re-suspend the cells in PBS at a concentration of about 750, 000 cells per milliliter. Next, mix ultra-low melting point agarose in PBS, and heat the mixture at 90 degrees Celsius, until the agarose melts.
Load the agarose mixture into a syringe, with an attached 0.22 micron filter. Place a syringe pump in front of a space heater set to 80 degrees Celsius and place the syringe in the pump. Fill a second syringe with the yeast suspension, and fill a third syringe with fluorinated oil, with 2%ionic fluorosurfactant.
Load both syringes into the syringe pumps. Connect the tubing from the syringes to device A.Place a 15-milliliter conical tube in an ice bucket, and guide the outlet tubing into the conical tube. Set the flow rate for each syringe, and collect approximately one milliliter of emulsion in the 15 milliliter conical tube.
After waiting five minutes for the agarose in the tube to set, add an equal volume of 20%perfluoro-octanol in fluorinated oil to the emulsion. Mix the emulsion and the perfluoro-octanol by inverting the conical tube a few times. Centrifuge the broken emulsion at 2, 000 times G for two minutes.
Be sure the hydrogels have pelleted above the oil and PFO phases. Remove the oil and PFO phases. Add two milliliters of tet(W)buffer to re-suspend the hydrogels.
Transfer the suspension into a new 15-milliliter conical tube. Centrifuge the tube again at 2, 000 times G for two minutes. Remove the supernatant, and re-suspend the hydrogels in tet(W)again.
After spinning down the hydrogels at 2, 000 times G for two minutes, re-suspend the hydrogels in two milliliters of yeast culture medium. Incubate the tube overnight at 30 degrees Celsius, with shaking. Each strains grow at different rates.
Choosing an appropriate media and incubation time is essential to ensuring that the yeast grow within the hydrogel, but do not overgrow, leading to cells escaping into the media. First, transfer the hydrogels to a 15-milliliter conical tube. Centrifuge the tube at 2, 000 times G for two minutes.
Wash the hydrogels twice with PBS and then once in spheroplasting buffer. Perform a 40x dilution of spheroplasting enzyme in spheroplasting buffer. Then, add one milliliter of the diluted enzyme to the hydrogels.
Incubate the tube of hydrogels at 37 degrees Celsius for one hour. The treated yeast will look more transparent. From the tube containing the hydrogel suspension, withdraw 0.8 milliliters of the suspension from the bottom of the tube, and transfer it to a one-milliliter uncapped syringe.
Place the syringe in the 3D-printed syringe holder. Centrifuge the syringe and holder at 2, 000 times G for two minutes. Before proceeding, ensure that the hydrogels are tightly packed at the bottom of the syringe.
Place 240, 000 drop-seq beads in a 15-milliliter conical tube. Centrifuge the tube at 1, 000 times G for one minute. Remove the supernatant, and re-suspend the beads in two milliliters of 0.9x yeast lysis buffer, with 500 millimolar sodium chloride.
Insert a stir bar, and transfer the bead suspension to a three-milliliter syringe. Prepare another syringe, containing several milliliters of PFPE-PEG surfactant in fluorinated oil. Obtain the previously prepared syringe of yeast clone packed hydrogels.
Evacuate the acquiesce head, and cap the syringe. Insert the three syringes, the hydrogels, the bead suspension, and the oil into syringe pumps. Connect the syringes via tubing to device B, the encapsulation device.
Place the end of the outlet tubing into a 50-milliliter conical tube on ice. Set the flow rate for each syringe. Collect approximately 1, 000 milliliters of emulsion, or run the device until there are no hydrogels remaining.
Then follow the drop-seq protocol for cDNA synthesis, library prep, and sequencing. Using a microfluidic device, yeast cells were encapsulated in 160-micrometer droplets. An eight-fold splitter divided these droplets into eight 60-micrometer droplets.
Overnight incubation resulted in isogenic yeast colonies growing within some of the hydrogels. Prior to loading the yeast hydrogels into the second microfluidic device, they were washed and immersed in a solution to digest the cell walls. Proper digestion was verified by microscopy, with treated yeast cells having a more reflective morphology.
A stream of mRNA capture beads in lysis buffer was mixed with a stream of close-pack yeast hydrogels prior to the drop-making junction of the second microfluidic device. In the resulting emulsion, about 10%of the droplets collected contained one bead with a lysed colony. This isogenic colony sequencing workflow was used to analyze the white opaque switching response of C.albicans.
Principal component, PC, analysis and a tSNE dimensionality reduction indicated general concordance between the sample data set and a reference data set. TSNE analysis revealed three clusters of cells. While cluster two was predominantly comprised of cells from the sample data set, clusters zero and one were comprised of cells from both samples.
Overlaying WH11 expression on the tSNE indicated that cluster one likely contained white colonies. STF2 expression increased in cluster one, consistent with previously obtained data. In clusters zero and two, WH11 and STF2 were significantly down-regulated compared with cluster one.
Androgen microbes have an ever-increasing potential to mass produce biologics for treating a wide variety of diseases. Following this procedure, one can apply a variety of bioinformatic tools to further analyze the sequencing data.
