hi my name is Mike horns I'm the chief
executive Donna might be a part of the
black trade group of companies we
specialize in a single cell droplet
biology applications and today this
video we mostly prevented by my
colleague Patrick Gilligan is a senior
application scientist that Donna my
buyer and today Patrick's going to be
talking to you about single-cell RNA see
this technology enables tens of
thousands of seen themselves to be
encapsulated along with bar-coded mrna
capture beams in droplets that are just
1/10 of a millimeter in diameter which
equates to about 100 people eaters in
volume and this enables sequencing of
tens of thousands of single cell
transcriptome so Patrick over to you so
two interesting things have happened in
recent healeth micro fluidics one is
there now off-the-shelf systems that you
can buy for micro fluidics which are
usable and ride on that that skilled
with molecular biologists can use them
the other exciting thing that's happened
is there now applications which are
really genuinely useful in a biology lab
for instance they are on AC single cell
application to give a little bit of
background on droplet microfluidics what
typically happens is he have a
microfluidic chip that makes aqueous
droplets in oil
these aqueous droplets on the size range
of about 100 Peter liters and then made
it rates of approximately thousands of
droppers per second typically you'll
have two flows such as cells and some
reaction links that have been mixed just
a few hundred 100 microns or so before
the junction they toast droplets and
made in flourish oils Ferris oils are
similar to hydrocarbon oils and see that
they have fluent atoms substituted for
hydrogen atoms Fleurus oils
effectively isolate the contents of one
acre strata from the other so they make
the droplets behave as discrete micro
reactors first oils
by compatible safe non-toxic and sells
can typically stay alive if necessary in
Flora's oil droplets for days another
thing that happens with droplet
microfluidics is when you lies a drop
when you lies a cell in a hundred pink
liter droplet the mRNA from Excel ends
up being in a higher fit of
concentration this makes such this makes
things like capture mRNA on mo- beings
why tpz are highly efficient because of
our high Victor concentrations in more
detail and their single cell in - seek
application you have three streams - AQ
streams one of which is the blanket of
beads and the other as it sells
bar-coded beads beads that have a
marionette capture oliver's on them and
part of it Aligarh sequence is a barcode
the barcode is synthesized such that in
free ala go on one bead has the same
backward sequence but every bead in the
library has different by code sequence
what this allows you to do is that after
Marilyn captured and reverse
transcription and sequencing you can
infer the cell of origin by clustering
cells by by code sequence on the
microfluidic chip the two streams with
the baguette of beads in themselves and
mixed approximately hundred microns
before the junction then what you're
aiming to do is to capture single cells
single bank red beans in droplets once
the cell gets into a droplet of a B the
lysis buffer braces Sullivan and the
mRNA is captured on the bead once
emotion comes off a chip you break the
emulsion wash the beans and then you go
straight into the reverse transcription
for the sequencing in this way the
droplet system is played poorly
compatible with downstream processing
steps to go into more detail bead is
decorated with many copies of a mulligan
delegate has a binding site for
sequencing primer and then it has the
bad guy sequence and it also has a poly
DT switch that captures they now name
and primes roots conscription this slide
gives you an overview of what the system
looks
at the top you can see a schematic of
what the microfluidic system looks like
on the right is the drawing of a chip
the chip has two separate microphone
exert cuts on it of what you only use
one at any given time at the bottom you
can see a close-up of the junction and
you can see it becoming in and the
center central stream and the cell will
appear and then downstream you can see
droplets so this is what the junction
looks like when it's running at 4,000
droplets per second which is a typical
proper production rate on the right of
droplets after that come off the chip
you can see be eaten approximately 10%
of the chocolate you can see the cells
because they've already been lies by the
lines discovered here we have a droplet
microfluidics system for there are three
pumps on for each of the three flowers
here we have the high speed digital
microscope which we developed
specifically for proper micro fluidics
for violin tea there's two reasons for
this one is that we in droplet
microfluidics ships are making profits
to drop the production rates are quite
high on the order of several thousand
dollars of settings their schematic
quite heart quite hard to see the
droppers under standard microscope the
other reason is the stage is
specifically built to handle a variety
of different microfluidic equipment in
this case is the single scattering C
chip unknown on the stage also the lens
we have on the scope is a long working
business lens which will be plenty of
working distance of different
microfluidic devices here we have a
sample book which we using for loading
the beads reason for this is that the
beads in this protocol of a large and
delicate and they're easily broken by
mainly experience for instance so this
is the gentle wave feeling of it
upstream of this of the sample of this
the sample injection valve attached the
supplementation goggles and fitting
through which you can inject the
extensive sample with business ended one
lower like syringe and here we have the
RNA seek chip only seek chippers Alvin
an interface with the connector
that's which is there and this is one of
our standard connectors it's made for
making microfluidic connections to chips
here you can see a video with RNA
sequencing shipped running you can see
it at beats coming in through the
central channel and the scales come in
from the side and drop asking for every
Junction for more information please
visit our dynamite micro fluidics
website a few more of our videos online
thank you