This protocol will enable the use of single-cell RNA-seq for initial phenotyping. And, when paired with single-cell ATAC-seq, the reconstruction of gene regulatory networks. The use of MULTI-seq keeps costs low for initial investigation, and fixing paired samples for single-cell RNA-seq and single-cell ATAC-seq allows for time course analysis of gene regulatory networks.
Investigators can utilize this protocol to understand the genetic networks controlling development and disease progression. To begin, add 20 microliters of anchor barcode solution to each sample and pipette it to mix. Incubate it on ice for five minutes.
Then add 20 microliters of co-anchor solution to each sample, and incubate on ice for another five minutes. Add one milliliter of ice cold 1%BSA and PBS to each sample and centrifuge the cells at 300 x g for five minutes at four degree Celsius. Remove the supernatant without disturbing the cell pellet.
Then add 400 microliters of ice cold 1%BSA and PBS. Centrifuge at 300 x g for five minutes at four degree Celsius. Determine the concentration of cells for each sample using a hemocytometer, and calculate the total number of cells and volume required for Gel Bead-in-Emulsion or GEM.
Combine the calculated volumes from each sample in a new sterile 1.5 milliliter tube, and determine the final cell concentration of the combined samples. After GEM generation and barcoding, perform gene expression cDNA cleanup using size selection, making sure not to discard the supernatant from the first round of selection, which contains the sample barcode. Transfer the supernatant to a new sterile 1.5 milliliter microcentrifuge tube and store it on ice.
Vortex the paramagnetic bead-based size selection reagent, and add 260 microliters of the reagent and 180 microliters of 100%Isopropanol to the supernatant. Pipette the mix 10 times and incubate at room temperature for five minutes. Place the tube on a magnetic rack and wait for the solution to clear.
Once the solution is clear, remove and discard the supernatant. Add 500 microliters of 80%ethanol to the beads for 30 seconds, then discard the supernatant. Repeat this for a total of two washes.
Briefly centrifuge the beads and return them to the magnetic rack. Set the timer to two minutes. Then remove the residual ethanol with a P10 pipette, and air dry the beads on the magnetic rack for the remaining two minutes.
Remove the beads from the magnetic rack and resuspend them in 100 microliters of elution buffer. Pipette thoroughly to resuspend, and incubate at room temperature for two minutes, then return the beads to the magnetic rack and wait for the solution to clear. Transfer the supernatant to a fresh 1.5 milliliter microcentrifuge tube.
Repeat the cleanup as demonstrated, using half the volume of elution buffer. For the samples destined for scATAC sequence, remove any liquid from the microcentrifuge tube using a P200 pipette, and flash-freeze the samples in ethanol dry ice. To dissociate the cells in the samples destined for scRNA sequencing, perform the dissociation steps using papain, as described in the text manuscript.
Centrifuge the cells at 300 x g for three minutes at room temperature, and remove the supernatant without disrupting the cell pellet. Add one milliliter of chilled PBS, and mix 10 times or until the cells are completely suspended. Again, centrifuge at 300 x g for five minutes at four degree Celsius, and repeat the wash with PBS twice.
Start vortexing the tube at the lowest speed setting and add 900 microliters of chilled methanol, drop by drop, while continuing to gently vortex the tube to prevent the cells from clumping. Incubate the cells on ice for 15 minutes. Determine the concentration of the fixed cells using a hemocytometer, and assess the efficacy of the fixation step using trypan blue.
Store the frozen tissue and fixed cells at minus 80 degrees Celsius. MULTI sequence barcoding enables the combined sequencing of multiple samples and their subsequent computational deconvolution. The sample of origin can be determined for each cell based on their barcode expression.
These combined samples can be analyzed as a single dataset for the purposes of cell clustering and cell type identification. Because each cell is barcoded before GEM generation, cell doublets will have a high probability of showing expression for multiple MULTI sequence barcodes, and a majority of doublets can therefore be identified and removed prior to clustering and cell type identification. The single-cell ATAC sequencing can be used to generate a dataset with cell types to match those found by single-cell RNA sequencing.
The pairing of single-cell RNA sequencing gene expression and single-cell ATAC sequencing DNA accessibility information enables the reconstruction of gene regulatory networks. When attempting the protocol, keep in mind that the two-minute timing of the ethnol evaporation is crucial for sequence retrieval in the subsequent elution steps. Reconstructing gene regulatory networks using paired single-cell RNA-seq and single-cell ATAC-seq produces targets for gene overexpression and knockout studies.
MULTI-seq can be employed at this stage to identify cell type specific effects.
