Transcriptomics enable us to gain a deep understanding of cellular program and their reaction towards external changes. However, despite a considerable reduction in library production and sequencing costs over the last decades, the application of this technology at the scale needed for drug screening is still unaffordable, hindering the great potential of these methods. Our study present a cost-effective system for transcript based drug screening.
Combining miniaturized perturbation cultures with minibug transcriptomics. The optimized minibug protocol provides informative biological signal at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. One of the key benefits of combining transcriptomics with unbiased drug screening is the potential to identify new drug targets that have not been previously considered.
Conventional drug screening approaches often focus on established target molecules or pathways, hindering the identification of new targets, and potentially resulting in drugs with unforeseen side effects and restricted effectiveness. To begin, thaw the PBMCs cryo vial in a water bath at 37 degrees Celsius for two to three minutes while gently inverting it. Then, transfer the thawed cells to a 50 milliliter conical tube.
Rinse the cryo vile with one milliliter of warm complete growth medium, and add the solution drop-wise to the cells in the conical tube to perform the first dilution step. Now, centrifuge the cell suspension for five minutes and tip the conical tube fluently to remove the supernatant. Re-suspend the cells in three milliliters of warm complete growth medium.
For cell counting, mix 10 microliters of cell suspension with Trypan blue. Count the cells using 10 microliters of stained cell solution, either with an automated cell counter or a cell chamber. Use warm complete growth medium to dilute the cells to achieve a final concentration of one times 10 to the six cells per milliliter.
Then seed 100 microliters of cell suspension into each well of a 96-well cell culture plate. Add 100 microliters of the two times drug dilution to each well. To harvest the cells, centrifuge the cell culture plate for 10 minutes.
Use a vacuum pump to remove the supernatant while placing the pump tip at the lower corner of the well to retain cells. Now add 200 microliters of ice cold PBS to each well. After centrifuging the plate for five minutes, remove the supernatant.
Then add 15 microliters of lysis buffer to each well. Using adhesive sealing film, seal the plate to guard against contamination. Vortex the plate and centrifuge it for one minute.
Finally, collect six microliters of the lysed cell solution in a PCR plate. To begin, harvest the drug treated PBMCs by centrifugation and collect the cell lysate in a PCR plate. Centrifuge the plate to collect the lysate at the bottom.
Next, perform mRNA denaturation using a thermocycler. Then add six microliters of the reverse transcriptase reaction mix to each well to achieve a total volume of 12 microliters. Seal the plate to guard against contamination and to prevent evaporation.
Centrifuge the plate after vortexing it briefly. Position the plate on the thermocycler and initiate the reverse transcription reaction program. After reverse transcription, centrifuge the PCR plate, add 15 microliters of the pre amplification mix into each well and seal it.
Place the sealed plate on the thermocycler and start the pre amplification reaction. To clean up the CDNA, add magnetic purification beads to each well and incubate for five minutes at room temperature. Position the PCR plate on a magnetic rack for five minutes or until the beads separate.
After that, carefully remove the supernatant. While maintaining the plate on the magnetic rack, gently introduce 100 microliters of freshly prepared 80%ethanol to cleanse the beads. After a 30-second incubation, use a pipette to eliminate the supernatant.
Allow the beads to air dry on the magnetic rack for five minutes or until the ethanol fully evaporates and the beads no longer appear shiny. After removing the plate from the magnetic rack, re-suspend the beads in 20 microliters of nuclease free water. Reposition the plate on the magnetic rack until the beads are separated.
Transfer the eluette to a fresh PCR plate prepared for CDNA quality control and tagmentation. For tagmentation, add three microliters of the tagmentation mix for each reaction onto a new PCR plate. Add one microliter of CDNA to every reaction.
Start the tagmentation reaction immediately. Then neutralize the reaction by adding one microliter of neutralized tagment buffer to each reaction. For enrichment PCR, add nine microliters of the enrichment PCR mix into each reaction to achieve a total volume of 14 microliters.
Start the enrichment PCR program. To begin, combine uniquely indexed libraries in an equimolar ratio. Measure the concentration of the final pool with a high sensitivity assay to calculate the sample molarity using this equation.
Now, load the flow cell following the specifications of the instrument and any experimental optimization. Select sequence on the screen to commence the run setup. Follow the onscreen instructions to load the flow cell sequence by synthesis cartridge, clustering cartridge, and buffer cartridge.
Upon recognition of all reagents by the equipment, add the run name in run setup. Specify the sequencing details as pair to end with both reads being 51 base pairs in length. Additionally, sequence two index reads of eight base pairs each.
Determine the output directory for data storage and tap on Review. After verifying the correctness of all sequencing parameters, select Start Run to commence sequencing.
