What is the channel depth of the microfluidics device?
The channel depth is 125 microns.
What size holes should I punch in the PDMS mold?
If you use the tubing listed in the protocol and paper, then you want to punch holes with a 0.75 mm biopsy punch.
I decided to make my own devices. Now I want to Aquapel treat them. How do I do that?
Here is a step-by-step guide for Aquapel treatment: Aquapel-Treatment-of-Drop-seq-Devices.pdf (5859 downloads)
Is there a company I can purchase devices from?
Devices can be purchased ready-made from Nanoshift, FlowJEM, or uFluidix. You can choose to have the devices pre-treated with Aquapel (or any other hydrophobic treatment) by the company, or do it yourself (see protocol above).
I bought beads from ChemGenes and I see damaged beads and/or debris.
We see this as well. It is likely that the process of oligonucleotide synthesis renders the beads more fragile/brittle. We also notice that if beads are stirred too aggressively during droplet generation, additional damage can occur. Most debris is removed during the process of harvesting the beads from droplets, presumably because the wash steps (with 6x SSC) remove lower density material.
I just made a device but the droplets seem smaller. Why? Does it matter?
Most likely, the channel depth of your devices is smaller. Smaller droplets are fine, as long as the beads flow consistently and evenly through the device. Remember that with smaller droplets, the cell and bead occupancy rates will also decrease (click here for more information).
Where can I get the required oligos, and what purification methods should I request?
We get our oligos from IDT (Integrated DNA Technologies). Only the TSO requires HPLC purification; standard desalting is sufficient for all the other oligos.
How much read-depth do I need for my cells?
The number of reads per cell you choose is dependent upon the scientific question being asked, and the size of the cells you are sequencing. For our retina dataset, we used a low read-depth (~13,000 reads per cell) because retinal cells have a low average diameter. We found additional read-depth to be helpful in our cell-cycle analysis of HEK and 3T3 cells, which are considerably larger than primary retinal cells.
Here is a saturation analysis of HEK and 3T3 cells that was included with the submitted manuscript but was removed from the final manuscript because of space considerations.
Where can I find the meta data used in the paper (Human/Mouse/Mixed)?