TITLE: BioMark Single Cell Protocol (Two-Step RTSTA)
AUTHORS: Shaina Robbins, Alison Moss, Sean Nieves

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol utilizes Fluidigm's Biomark system which performs high-throughput real-time PCR that can assay 48 or 96 genes for 48 or 96 samples respectfully. This protocol is used for gene expression targeting samples at the single-cell scale and can be used with the 48.48 Dynamic Array integrated fluidic circuit (IFC) or the 96.96 Dynamic Array IFC. Note that this protocol assumes that single-cell samples are captured using the Arcturus Laser Capture Microdissection system.</div></div>

[STEPS]
?. [LCM Extraction]
Prepare lysis buffer as follows:Lysis enhancer (CellDirect kit)Resuspension buffer (CellDirect kit)Total lysis buffer per sample
0.5 µl
5 µl
5.5 µl
?. [RNA Dilution Series (Optional)]
Prepare total RNA dilutions of 1 ng/ul, 300 pg/ul, 100 pg/ul, 30 pg/ul, 10 pg/ul, 3 pg/ul, 1 pg/ul, 300 fg/ul and H2O.Add  of RNA to lysis buffer.Treat RNA dilution samples the same as the LCM samples.
1 µl
5.5 µl
We assume the average RNA yield from single cell LCM is approximately 10 pg.
?. [Reverse Transcription]
Transfer all samples to a PCR plate.Add 5X VILO Reaction Mix to each cell lysate (including dilution series).
1.2 µl
VILO Reaction Mix is part of the VILO cDNA synthesis kit and always runs short. Make sure sufficient reagent is available before RT.
?. [LCM Extraction]
Capture single cells on CapSure HS caps.Add  lysis buffer per capIncubate at for  cap-side downIncubate on ice for cap-side downIncubate on ice for right-side upSpin down and transfer lysate to PCR tube or plate or freeze at
5.5 µl
[on heat block]
-80 °C
Sample size will vary, i.e., single cell, 10-cell pool, 100-cell pool, etc.
?. [Reverse Transcription]
Incubate plate at  for , snap cool on ice and spin down.
65 °C
?. [Reverse Transcription]
Mix the following reagents and add  to each sample (for 96.96 Biomark chip):10X Superscript III Mix (VILO kit)  x 110 = T4 Gene 32 Protein  x 110 = H2O  x 110 = Final sample volume including lysate =
1 µl
0.15 µl
16.5 µl
0.12 µl
13.2 µl
0.73 µl
80.3 µl
7.5 µl
?. [Reverse Transcription]
Incubate PCR plate using the following thermocycles:forforforforforHold at
25 °C
50 °C
55 °C
60 °C
70 °C
4 °C
?. [Preparing Pre-Amp Primer Pool]
For 96.96 chip, each primer pair is diluted to 50 uMPrimer pairs (50 uM) x 96 = H2O Total = of primer pool (500 nM concentration)
2 µl
192 µl
8 µl
200 µl
?. [PreAmp]
Mix together and add  to each RT product (for 96.96 chip):Taqman PreAmp Master Mix x 110 = 500 nM primer pool x 110 = Final sample volume =
10.5 µl
9 µl
990 µl
1.5 µl
165 µl
18 µl
?. [PreAmp]
Incubate PCR plate using the following thermocycles:for 22 cycles of:for for
95 °C
96 °C
60 °C
We consider 22 cycles of pre-amp optimal for LCM captured neurons. Number of cycles can be adjusted acccording to sample type.
?. [PreAmp]
Exonuclease TreatmentMix the following and add to each sample (for 96.96 chip):Exonuclease I reaction buffer 10X  x 110 = Exonuclease I  x 110 = H2O x 110 = Final sample volume =
6 µl
0.6 µl
66 µl
1.2 µl
132 µl
4.2 µl
462 µl
24 µl
?. [PreAmp]
Incubate PCR plate using the following thermocycles:for for Add of TE buffer to each reaction.
37 °C
80 °C
51 µl
Water can be used if long term storage of amplified cDNA is not required. Samples can be stored in  if they will not be taken to the next step right away.
?. [Prepare Sample Pre-Mix and Samples]
Prepare the sample pre-mix:2X SsoFastEvaGreen Supermix with low ROX 20X DNA Binding Dye Sample Loading Reagent (green cap) Add sample pre-mix to  of sample after PreAmp for a total of per sample.Vortex the Sample Mix solution for a minimum of , and centrifuge for at least .Prepared reactions can be stored for short times at until the samples are ready to be loaded into the chip.
4 µl
0.4 µl
4.4 µl
3.6 µl
8 µl
4 °C
?. [Prepare Assay Mix]
Combine the following:2X Assay Loading Reagent (yellow cap) 20 uM primers Vortex the Assay Mix for a minimum of and centrifuge for at least to spin down all the components.
The same preparation of primers can be used for the Fluidigm 48.48 Dynamic Array IFC and the Fluidigm 96.96 Dynamic Array IFC.
4 µl
4 µl
Vortex thoroughly and centrifuge all samples and assay soltions before pipetting into the chip inlets. Failure to do so may result in a decrease in data quality.The final concentration of each primer is 5 uM in the inlet, and 500 nM in the reaction chamber.
?. [Priming and Loading the Dynamic Array IFC]
Inject control line fluid into each accumulator on the chip (see diagram).Remove and discard the blue protective film from the bottom of the chip.Place the chip in the IFC Controller MX for the 48.48 chip or the IFC Controller HX for the 96.96 chip, then run the Prime (113x) script for the 48.48 chip or the Prime (136x) script for the 96.96 chip.When the prime script has finished, press Eject to remove the primed chip from the IFC Controller. Pipette  of each assay and  of each sample into their respective inlets on the chip (refer to diagram).IMPORTANT: You must pop/remove any air bubbles that are in the inlets. This must be done in under  .Return the the chip to the IFC Controller.Using the software, run the Load Mix (113x) script for the 48.48 chip or Load Mix (136x) script for the 96.96 chip to load the samples and assays into the chip.When the Load Mix script has finished, remove the loaded chip from the IFC Controller.Remove any dust particles or debris from the chip surface using scotch tape. you are now ready for the chip run.Pipette of each assay and of each sample into their respective inlets on the chip (refer to diagram).IMPORTANT: You must pop/remove any air bubbles that are in the inlets. This must be done in under .Return the the chip to the IFC Controller.Using the software, run the Load Mix (113x) script for the 48.48 chip or Load Mix (136x) script for the 96.96 chip to load the samples and assays into the chip.When the Load Mix script has finished, remove the loaded chip from the IFC Controller.Remove any dust particles or debris from the chip surface using scotch tape. you are now ready for the chip run.
CAUTION! Due to different accumulator volumes, use the appropriate control syringe for your chip type:  for the 48.48 chip or  for the 96.96 chip.
CAUTION! While pipetting, do not go past the first stop on the pipette. Doing so may introduce air bubbles into the inlets.
5 µl
5 µl
?. [BioMark Run]
Double-click the Data Collection Software Icon on the desktop to launch the software.Click Start a New Run.Check the staus bar to verify the lamp and the camera are ready. Make sure both are green before proceeding.Place the chip into the reader.Click Load.Verify chip barcode and chip type. Choose project settings (if applicable). Click Next.Chip run file: Select New. Browse to a file location for data storage. Click Next.Application, Reference, Probes: Select Aplication Type--Gene Expression for version 3.1.2 or higher software (for earlier versions, contact Fluidigm Technical Support). Select Passive References- ROX. Select Probe--Single probe. Select probe type- EvaGreen. Click Next.Click Browse to find the thermal cycling protocol file. For BioMark HD- GE Fast 48x48 PCR+Metl v2.pcl or GE Fast 96x96 PCR+Melt v2.pcl. For BioMark- GE 48x48 PCR+Metl v2.pcl or GE 96x96 PCR+Melt v2.pcl.Confirm Auto Exposure is selected.Click Next.Verify the chip run information.Click Start Run.