(upbeat music) - [Mason] Hello, and
welcome to this episode of our Active Motif webinar series. My name is Mason Brooks and today, we'll be hearing from my
colleague, Dr. Joshua Messinger who is an R&D scientist
here, at Active Motif. Today he will be talking about strategies for enabling epigenetic data to be used, not just in research, but
in the clinic as well. - [Joshua] Hi, Mason. Thank you so much for that
wonderful introduction. So I am here today to talk about a high-throughput tissue
pr
ocessing pipeline that I've been working on here
at Active Motif, in the lab. Really trying to get the
speed up for processing mammalian tissues into
usable chromatin samples that can be used for ChIP-seq. We try and make epigenetics
a bit little more amenable to clinical medicine,
translational medicine, and personalized therapeutics. So, I'm sure myself, and
the rest of Active Motif, and hopefully everyone watching out there, is a really big epigenetics enthusiast. And I'm sure that we're all
interested in epigenetics from a basic science,
biochemical perspective, but also, I'm sure we can all appreciate that there is tremendous
potential for epigenetics, and being toggleable in
the clinical setting. So, this was an image that I found from a recent review in 2017, showing how epigenetics could be targeted in cardiovascular diseases. But a lot of these principles
hold true for really, any disease state. So we can see here, from this image that we can target DNA
methylation with DNMT i
nhibitors, we can target histone modifications with HDAC or histone deacetylases or histone acetyltransferase inhibitors which have broad effects
on gene transcription. So in this way, we're
really able to target what gene expression looks like
in these disease state cells targeting their epigenetic regulators. And in this way, we can
have a much wider impact on what the transcriptional
network is doing in these disease states, and hopefully, it will be to work targeted and improved therapeutic
outcome. However despite all of
these possibilities, clinical implementation of epigenetic data has been really slow and I'm sure a lot of us have
been on the ENCODE website and have looked at epigenetic data that gets published often
in high impact journals. And seeing as there's a wide availability of epigenomics data, the personalized medical therapeutics have been really slow to
implement in the clinics targeting epigenetic regulators. And, well why is that? First of all, there's sometimes v
ery
limited clinical samples. A lot of these disease states, you need to get large numbers to make conclusions on a population level, and you can only get so
much tissue or material from vulnerable populations
and as a result, there's often not enough material to answer relevant questions. Oftentimes, you only get a couple mg's or maybe a gram of tissue, you process that into a chromatin sample and you realize upon your input prep that you're not gonna have enough to do a ChIP-seq experiment. An
d in that case, you can't
actually ask any questions that are gonna be relevant to figuring out what might be targetable
in these disease states. It's also extremely
difficult to make chromatin. I'm sure a lot of people
out there would agree that a chromatin prep is cumbersome, it can take a lot of time and as a result, you need an
army of epigenetic scientists constantly making chromatin
from these samples. And often, these chromatin samples then need to be sonicated for ChIP and if you have in
consistent
shearing of your samples, you could end up having to throw
away entire chromatin preps just because they're not the right size. And I know that inconsistency in shearing has been a major issue
for epigenetic scientists. So we need to find a way to
standardize this shearing, and know that each time we
sonicate a chromatin sample, we get high quality, good
DNA of the right size, that is amenable for ChIP. And lastly, I wanted to point out that there are oftentimes barriers
in tissue hom
ogenization. We are intact organisms and our tissues do not want to become
single cell suspensions. And for the most part,
that's a really good thing because it keeps us alive. But from a chromatin material perspective, this can be a major issue because if we can't make a single celled suspension of the tissue, we have a low yield of cells, there's low amounts of DNA, and we circle on the first point, where now, there's not enough material to answer the relevant questions. So all of these issues
become major problems in terms of implementing epigenetics into the clinical space. And part of this project is hoping to standardize a pipeline that makes clinical
tissue processing faster, more standardized, and
consistently reliable so that we can ask high
impact epigenetic questions and understand how we can more accurately target these disease states. So, how do we bring epigenetics from the bench to the bedside? That's the major focus here. And we need a high-throughput,
consistent method
for processing mammalian
tissues for chromatin for epigenetics studies. We need to know that we can do it quickly, the first time, and correctly every time so that we can make the most
of these precious samples. So one of the techniques
that I've been playing with here in the lab, is called a rapid bead
based homogenization. And for this, I've been
using a machine called the Omni Bead Ruptor, which is a high speed, bead
based homogenization machine, capable of generating
single cell suspensions
from a number of different
mammalian tissues. And for these initial experiments, I did these in mouse tissues. So, I tested the capabilities of three different homogenization buffers to homogenize this tissue, two different bead sizes, I used a 2.4 mm and a 5 mm bead, which you'll see the data
for, in just a little bit. And I did this is seven mouse tissues. I did this in spleen, kidney,
liver, brain, heart, lung, and colon. And for the sake of brevity today, I'm really just gonna be zooming in
on the data
that's been generated from mouse spleen. So first, I just wanna show everyone that the bead-beating can yield a homogenous single cell suspension, and this is mouse spleen. So what we're looking at here is, this is a bright field image
of trypan stained single cells, generated from bead
homogenizing mouse spleen. I know it can look a little weird, there's a lot of gray dots so I just kinda wanna draw
a circle around the region. All of the gray dots in
that region are either single c
ells or intact nuclei. At this point, it is a
little bit difficult to tell whether we have a single cell suspension or we have a nuclear isolation because there's no un-homogenized control that I can really put on a hemocytometer so we have to do some further studies to figure out exactly what
these membrane particles are. But in some data that I'm not showing, you can spin down these samples
and do a buffer exchange, and you don't lose any genetic material, implying that this homogenization pro
cess does not lyse the cell
and release nucleic acid that gets lost in the
homogenization buffer. However, one of the buffers that the homogenization takes place in, is the same homogenization buffer, which I'll show you in just a second. So in theory, all of these samples can go from homogenization to
sonication in the same step, with no spin step required. So now that we had generated
the single cell suspensions, I wanted to sonicate the material to generate usable material
for a ChIP-seq expe
riment. So to sonicate the material, I've been using the PIXUL
Multi-Sample Sonicator. Our PIXUL Sonicator is a new
product here, at Active Motif that we have been extremely excited about. And it is a simple, rapid,
and extremely consistent method for shearing chromatin,
DNA, RNA, and protein. So if we look here, this
is the PIXUL instrument. It's actually a really
nice sized instrument, it fits right on a desktop. It's very user friendly in that it has a touchscreen and all the parameters are
e
asily set and visible for you. So just to highlight a little
bit what the PIXUL does, it is a 96-well plate based sonicator. So this uses U-bottom 96-well plates to sonicate materials. So in theory, you can do 96 samples, all in the same run which is really amenable to
clinical translational medicine, as they will need to process a large number of samples, simultaneously to generate the material
necessary for their experiments. It is a consistent and
high-throughput method for shearing of chroma
tin. You can do multiple parameters in one run. Each column in the plate
is a 96-well plate, so there are 12 columns. Each column is controlled by
its own pair of conductors, which means that each column
can run on its own protocol. So in this way, you can do chromatin shearing
optimization experiments in a high-throughput method, as well as sonicate samples that require different
sonication conditions, all at the same time by just putting them in different columns. It is extremely inexpensive,
in terms of the consumables that it uses. It uses a basic, standard
tissue culture, 96-well plate, which many labs out there
have large boxes of. So it's not expensive to run PIXUL. And I just wanna point out one more time that it allows for sonication of clinically relevant numbers
of samples, at one time. And this is the most exciting part, that it can sonicate so
many different samples under so many different conditions, consistent and reliably, all at one time. And I'll show you some
of the
data from that in just a second. Before we go in, I thought
it would be a good time to talk about what sonication actually is. So sonication basically takes place when we pulse these compression
waves through a sample, and these waves will cause bubbles to form that will expand over time. And as they reach a terminal
size, they will implode, and that releases a large amount of heat, as well as mechanical force, which then goes on to shear
and break up the nucleic acids. So this is really what ta
kes place when we sonicate samples. So now, just to show some of the results from what happens when I sonicated that single cell suspension
generated in mouse spleen. And what we're looking at here, is this is TapeStation data of the result of DNA that was purified from the input chromatin. And we're looking at three
different homogenization buffers. Buffer one, buffer two, and buffer three. This buffer two is the same
buffer that is used on PIXUL. So these samples are immediately
ready for soni
cation, for homogenization. And you can see that I used
a 2.4 mm and a 5 mm bead. And when we look at this column, this row here for PIXUL, this indicates whether the sample underwent sonication, or not. So N for no PIXUL sonication,
Y for yes, sonicated. And what I hope you can appreciate is that in all of the unsonicated samples, we don't see a migration of
small molecular weight DNA, like, around 300 base pairs, like we do in the sonicated samples, indicating that the PIXUL sonication is spec
ific for shearing that DNA. And we can see that in
all conditions tested, we generate a ChIP DNA
of about 300 base pairs, which is ideal for proceeding
downstream ChIP applications. So this was super exciting. All of the samples were
homogenized at the same time on the Omni Bead Ruptor. In one ml of homogenization buffer, in these 2 ml tubes that
are provided by Omni, at 4 m/sec, for 20 seconds. So just to backpedal for a second, all of this material was
homogenized in 20 seconds which is extrem
ely high-throughput, and basically means that multiple samples of mammalian tissues can be processed inside of 20 seconds, and then sonicated on PIXUL. Meaning that inside the same afternoon, you can generate high quality chromatin, a number of different mammalian tissues, all at one time. If we look at the input DNA concentrations from these preps, we're able to see that
just about every condition generates high concentration DNA of about 100 ng/mL. Some do better, and some
do a little bit wors
e. This data will need some replicates to understand exactly what
the range is in there. But in all conditions tested, there's enough to do at least a 5 or 10 microgram ChIP-seq
reaction in these samples, which is ample material for us saying where transcription factors and histone modifications
are within the genome. If we look at how pure this DNA is, most of it is very, very,
very pure, and very clean. We can see that there are
260/280 ratios of about 1.8, which is ideal for downstream next g
eneration sequencing applications. And if we quantify the percent of material that's actually sonicated, I hope you can appreciate is that about 95% of the material, is fully sonicated. And I'm drawing attention
to the 70% mark, right here, as this is what we use internally as a QC metric for good chromatin. If you're gonna use your
chromatin for a ChIP-seq reaction, we want it to be greater
than 70% sonicated. And in all conditions tested, all sonicated material, is greater than 70%, implying t
hat this is very,
very high quality chromatin. So having been able to generate
such high quality chromatin, or would appear to be so, I performed H3K9 acetyl ChIP-seq. So this experiment was conducted using our H3K9 acetyl antibody which is a wonderful antibody that does really robust ChIP-seq. And all I'm gonna show here so far, is what the NGS Library preps look like, from these reactions. So if we take a look here
at the TapeStation data of what these H3K9 acetyl ChIP-seq NGS Libraries look l
ike. We can see that in the, this is, by the way, all from the 5 mm bead homogenized sample. We can see that in all three homogenous agent buffers tested, the input material generates
a nice ChIP-seq library, it generates a nice input library at about 450 to 500 base pairs, which is what we expect for the kit that we used to generate these libraries. And the H3K9 acetyl ChIP reactions also generated very nice NGS libraries, averaging at about 450 to 500 base pairs, implying that DNA was isolated
during this ChIP reaction and it was readily converted into a next generation sequencing library. Because I had mentioned at
the beginning of this webinar that I had done this in
so many different tissues, I wanted to show that
the ChIP-seq libraries generated in these tissues
are all consistent. So we're looking at ChIP-seq libraries from H3K9 acetyl ChIP in kidney,
spleen, liver, and brain. And we can see that in all
three homogenization buffers, we get these really
nice ChIP-seq libraries av
eraging between 400 and 500 base pairs. And the same is also true for ChIP-seq that was done in heart, lung, and colon. So at this point, this process of 4 m/sec for 20 seconds homogenization
with a 5 mm bead and subsequent PIXUL sonication, represents a new high through-put method of homogenizing mammalian tissues that leads to high quality chromatin that can be used in ChIP-seq applications. These libraries are
currently being sequenced so we're very excited to see
what data comes out of this,
and then hopefully apply
this process to human tissues so that we can really start to answer some clinically relevant
and interesting questions and develop new targeted
personalized therapeutics for, in the epigenetic
space, or help labs do that. So just to conclude
what I've shown so far, beadruptor homogenization
represents a high-throughput way to generate single cell
suspensions of tissues. As I said before, all of
the single cell suspensions were generated in 20 seconds, and all of the sam
ples were sonicated on the same plate, in PIXUL, meaning that much of this work can take place inside of an afternoon. I, personally, have processed
up to three or four tissues inside of the same day, which is very, very quick. PIXUL sonication of resulting
single cell suspensions generates ChIP-ready high yield chromatin of consistent size. And I think that's the
most important part. That we have the capacity now to take these precious tissues and ensure that every time
we do a chromatin prep,
we get high yield, and a high quality that is worth answering
interesting questions in the epigenetic space. And that PIXUL sonicated tissue
chromatin is ChIP compatible and generates high quality NGS libraries. We still need to see how
these libraries sequence, but I'm very optimistic that we're gonna uncover some nice data and expand this pipeline
to human clinical samples. So with that, I just wanna thank everyone
for their attention. I really appreciate you
tuning into this webinar, giving m
e a chance to talk
about the exciting work that we have going on
here at Active Motif. A number of interesting products are coming out to support the
PIXUL Multi-Well Sonicator. So please take a look on our website. If you are interested in any high through-put epigenetic kits that are PIXUL compatible, please take a look at www.activemotif.com and feel free to contact me at
tech_service@activemotif.com. I'm always excited to answer questions about any of the interesting science that's going on
here in the lab, as well as just in the
general epigenetic space. So please don't hesitate to reach out if you have any questions. And thank you so much for your time today!
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