User Guide
Quick-Neuron™ Sensory - mRNA Kit (Small)
Catalog Number: SS-mRNA-S

Introduction
The Quick-Neuron™ Sensory - mRNA Kit (Small) facilitates rapid and efficient differentiation of human iPS or ES cells into a
population of sensory neurons in just 10 days. Our proprietary transcription factor-based stem cell differentiation method
uses synthetic mRNAs to produce highly pure populations of neurons without a genetic footprint. Quick-Neuron™ Sensory
differentiated cell cultures display typical neurite outgrowth and express a variety of neuronal markers, such as tubulin beta
3 class III (TUBB3) and a variety of sensory neuron markers such as sodium voltage-gated channel alpha subunit 9(SCN9A/Nav1.7), peripherin (PRPH), islet-1 (ISL1), and brain-specific homeobox/POU domain protein 3A(BRN3A/POU4F1). When handled and maintained according to the instructions in this user guide, sensory neurons are
viable long-term and are suitable for a variety of characterization and neurotoxicity assays.
Scale:The Quick-Neuron™ Sensory - mRNA Kit (Small) contains a set of reagents for use with a total of 4
wells of a 24-well plate.

Related Products:

Quick-Neuron™ Sensory - mRNA Kit (Large), Catalog Number: SS-mRNA-L
Quick-Neuron™ Sensory - Human iPSC-derived Neurons, Catalog Number: SS-mRNA-CW
Quick-Neuron™ Sensory - Maintenance Medium, Catalog Number: SS-MM

Contents
Upon receipt, store the reagents at the temperatures indicated in the table below. All reagents are shipped on dry ice.
Contents

VolumeStorageThaw

QNS-mRNA-P

4 x 9 μl

-80°C

On iceComponent N

840 μl

-20°C or -80°C

On ice or 4°C

Component S1

22 μl-20°C or -80°C

On ice or 4°C

Component P

50 μl-20°C or -80°C

Room temperature

Coating Material A

15.7 μl-20°C or -80°C

On ice or 4°C

This kit contains iMatrix-511 silk (Nippi, Inc.)

Condition of Use
This product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: August 03, 2023

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1Required Consumables
Item

VendorCatalog Number

24-well tissue-culture-treated polystyrene plate (e.g.,
Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-740

Lipofectamine MessengerMAX

ThermoFisher

LMRNA001

Opti-MEM I Reduced Serum Medium

ThermoFisher

31985062

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

Elixirgen Scientific
TaKaRaThermoFisher

ASB04-C, or
AK02N, orA3349401

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

0.01% Poly-L-Ornithine

Sigma-Aldrich

P4957-50ML

Extracellular Matrix such as
Laminin Mouse Protein, Natural, or
Geltrex Basement Membrane Matrix

ThermoFisher

23017015 orA15696-01

Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

InvivoGen

ant-pr-1

VWR

89167-106

StemFit Basic04 Complete Type, or
StemFit AK02N, or
StemFlex Medium

Puromycin (10 mg/ml)
12-mm glass coverslips**
100% Ethanol

Multiple Vendors* PBS should be used at room temperature unless otherwise specified.
** Glass coverslips from different brands might have different effects on the growth of neural cells. We recommend using glass
coverslips from Chemglass for the best results.

Source hPSC Culture Conditions
The Quick-Neuron™ Sensory - mRNA Kit (Small) gives the best differentiation results when source human pluripotent stem
cells (hPSCs) have been maintained in StemFit® Basic04, StemFit® AK02N, StemFlex™ Medium, or other similar culture
media which enable the maintenance of cultures by single-cell passaging. This protocol also assumes that the source
hPSCs are cultured in a 35-mm culture dish or one well of a 6-well plate. If iMatrix-511 silk is routinely used as a coating
substrate, prepare one culture dish or well precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS for this kit.
● The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04-C, NI511S).
● Differentiation should not be performed until the cells are at least 14 days post-thaw.

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●
●

We recommend preparing a minimum of 1.5 x 106 viable hPSC for use with this kit. This is usually obtained by using
1 well of a 6-well plate at 50-70% confluency.
For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

Drug Selection
Users should perform a puromycin kill curve for their cells to determine the minimum concentration required to kill all
non-treated cells within ~60 hours. Based on Elixirgen Scientific’s internal tests, the appropriate concentration ranges
between 0.5 and 2 µg/ml. We recommend maintaining 2 wells of untransfected iPSC (with standard StemFit conditions),
alongside the transfected wells, until after puromycin selection is performed. Treat 1 of those wells with puromycin at yourselected concentration so as to confirm that the puromycin is effective at killing the untransfected cells in your experiment.

Workflow
Note: This protocol assumes that Day 0 is a Monday.

* From Day 10, users may maintain differentiated neurons in the medium best suited for their needs, though we recommend
Quick-Neuron™ Sensory - Maintenance Medium, Catalog Number: SS-MM.

Preparation
Important Note! For the best possible delivery of QNS-mRNA-P into cells, we recommend Lipofectamine MessengerMax.
If users prefer another transfection reagent, please make sure that the reagent provides a transfection efficiency of ≥80%
prior to using this kit. QNS-mRNA-P mixed with Lipofectamine MessengerMax must be immediately applied to cultures andcannot be stored.
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as iROCK, can be stored at -20°C.
StemFit Basic04 Complete Type (Medium S)*
1. Thaw StemFit Basic04 Complete Type bottle overnight or multiple nights at 4°C.
2. Make aliquots of a convenient volume (e.g., 40 ml).
3. This solution, hereafter referred to as Medium S, can be stored at -80°C. Once thawed, Medium S should be stored
at 4°C for up to 2 weeks.
○ After thawing users may choose to add Penicillin-Streptomycin at a 1:200 dilution (e.g., 200 μl in 40 ml ofMedium S) before using Medium S.
*Medium S can be substituted with StemFit AK02N or StemFlex.

0.5X TrypLE Select with EDTA (Solution D1)
1. Mix 1 ml TrypLE Select Enzyme (1X) with 1 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.

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0.002% Poly-L-Ornithine solution (ornithine)
1. Take 700 μl 0.01% Poly-L-Ornithine solution and mix it with 2.8 ml PBS.
2. The 0.002% Poly-L-Ornithine solution, hereafter referred to as ornithine, can be stored at 4°C for up to 2 weeks.
1 mg/ml laminin stock solution (laminin)
1. Thaw Laminin Mouse Protein, Natural and chill PBS at 4°C or on ice.
2. Mix the Laminin Mouse Protein, Natural and PBS to make the 1 mg/ml stock solution, hereafter referred to aslaminin.
○ Laminin concentration varies by lot, so use the number specified on the vial or CoA when making yourcalculations.
3. Make aliquots of a convenient volume (e.g., 35 μl) and store at -20°C.
Medium N(S1)1.

Prepare Medium N(S1) using the reagents listed in the table below.
○ Thaw Components N and S1 for 20-30 minutes at the temperatures indicated in the “Contents” table on
page 1.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin all tubes down before use.
○ Keep Medium N(S1), and any subsequent media made with it, protected from light.
○ Store Medium N(S1) for up to 2 weeks at 4°C.
○ Leftover Components N and S1 can be discarded or saved at 4°C for up to two weeks.
Reagents

VolumeDMEM/F12

9.5 mlNeurobasal

9.5 mlGlutaMAX

100 μlPenicillin-Streptomycin (10000 units/ml; 100x)

200 μlComponent N

620 μlComponent S1

20 μl

Day -3~1 hour

Note: This protocol assumes that Day 0 is a Monday so Day -3 is Friday.
Plate Preparation1.

Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
○ Thaw Coating Material A for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Keep Coating Material A on ice.
○ Make sure chilled PBS is used for this mixture.
Reagents

VolumeCoating Material A

11.6 µl

Chilled PBS

3.5 mlelixirgensci.com

4

2. Add 400 µl diluted Coating Material A to each new well of 8 wells.
3. Incubate the plate at 4°C.
Note: For best results we recommend precoating the plate 1 day or up to a week before use and keeping at 4°C.
Alternatively plates can be precoated on Day 0 and placed at 37°C for at least 2 hours before use.

Day 0

~4 hours

Note: This protocol assumes that Day 0 is a Monday.
Plating1.

Prepare Medium iS by mixing together the following components in a 15 ml conical tube.
○ Warm Medium S, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium S should be stored at 4°C for later use.
Reagents

VolumeMedium S

5.5 mliROCK

5.5 µl

2. Aspirate old medium from hPSC culture and add 2 ml PBS.
3. Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
Keep the rest of Solution D1 at 4°C for use on Day 4.
4. Incubate the culture plate at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope,
continue to incubate at 37°C, 5% CO2 in 1-2 minute increments (see images below).

5.
6.
7.
8.

Carefully pipet out Solution D1 from the culture and add 1 ml Medium iS to the well.
Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
Using the same pipet tip, collect the cell suspension in a 1.5 ml tube.
Count cells and determine viability.

IMPORTANT! In this protocol, users will plate the hPSCs into 8 wells with 500 μl Medium iS per well. However, if users
know the best plating density already, prepare a cell suspension enough to plate 5 wells (4 wells plus 1 extra to accountfor the pipetting/plating error). We recommend aiming for 50-70% initial cell confluency on Day 1, with 20-50 cells percolony. Our data indicate that cell counts ranging from 1.0 - 2.4 x 105 viable cells per well are suitable. Cell count mayvary based on cell health and the method used for cell counting.
If the confluency on Day 1 is
• above the target range, the differentiation efficiency will decrease.
• below the target range, more cell death will be observed.
• For first time users, we recommend plating the following numbers of cells into each of 8 wells: 1.0, 1.2, 1.4,
1.6, 1.8, 2.0, 2.2 and 2.4 x 105 cells. On Day 1, 4 wells showing 50-70% confluency should be selected fortransfection.

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9. Take out the volume of the cell suspension needed for each cell density, according to the note above, and placeeach in a new tube labeled with the corresponding density.
10. Bring the volume of the cell suspension in each tube up to 500 µl with Medium iS.
○ If the volume in the tube exceeds 500 µl, centrifuge the required volume of cell suspension at 200 x g for 4
minutes, remove the supernatant, and resuspend the pellet in 500 μl Medium iS.
11. Aspirate diluted Coating Material A from each coated well and add 500 µl cell suspension to each well.
○ Most of the diluted Coating Material A should be aspirated but not completely to prevent the coated wellfrom drying before adding the cell suspension. The cell suspension should be added to the wellimmediately after diluted Coating Material A is removed. Handle one well after another.
12. Leave the plate flat at room temperature for 10 minutes.
13. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 1~8 hours

IMPORTANT! Observe all wells under a microscope and select any 4 wells that show 50-70% confluency for transfection
with QNS-mRNA-P. If none of the wells fall within the range of confluence, do not proceed.
First Treatment
1.

Thaw 1 vial of QNS-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium S at room temperature for
20-30 minutes.
2. Prepare QNS by the following steps:○ Tap the QNS-mRNA-P tube 3 times and then briefly spin it down before use.
○ Prepare a 15 ml tube and a 1.5 ml tube with 220 μl Opti-MEM each. Label the 15 ml tube “Mix 1” and the 1.5
ml tube “Mix 2”.
○ Add 4.5 μl Lipofectamine MessengerMax (LMM) to the Mix 1 tube and mix by brief vortexing. Leave it atroom temperature for 10 minutes (Mix 1). Keep the rest of LMM at 4°C for later treatments.
○ IMPORTANT! Immediately before 10 minutes pass (i.e., around 8 minutes), add 9 μl QNS-mRNA-P to theother 1.5 ml tube with Opti-MEM (Mix 2). Mix by tapping 5 times. Do not vortex.
○ 10 minutes after mixing LMM with Opti-MEM, add Mix 2 into Mix 1, and pipet up and down 8-10 times. This
mixture is called QNS. Leave QNS at room temperature for 5 minutes and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

220 µl

Opti-MEM

220 µlLMM

4.5 µl

QNS-mRNA-P~9 µl

3. Add 1.75 ml Medium S to QNS (for a final volume of 2.2 ml) and pipet up and down 2-3 times to mix.
4. Working with up to 2 wells at a time, aspirate the old medium out and add 500 μl of QNS mixture to each well.
Repeat until QNS mixture has been added to all wells.
5. Incubate the culture plate at 37°C, 5% CO2 for 2.5 hours.
Second Treatment
1. Pipet out the medium from each well and add 500 μl Medium S.
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QNS-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section.
Medium Change and Drug Selection
1.

Transfer 2 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
2. Pipet out the medium from each well and add 500 μl Medium N(S1) with puromycin.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.
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6Day 2~8 hours

IMPORTANT! Observe the QNS-treated cultures to make sure that they are reaching confluency (≥90%). If the cultures are<50% confluent and show signs of cell death (e.g., many floating cells), users should skip the third and fourth treatments on
Day 2 and proceed directly to the “Medium Change” section. First time users, who have plated cells at different densities,
should proceed to the third and fourth treatments for only two wells with higher plating densities. For the other two wells(with lower densities), skip the third and fourth treatments on Day 2 and proceed directly to the “Medium Change” section.
Third Treatment
1.

Thaw 1 vial of QNS-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium N(S1) at room temperaturefor 20-30 minutes.
2. Repeat Steps 2-5 of the previous “First Treatment” section using Medium N(S1) in place of Medium S to prepare
QNS mixture.
Fourth Treatment
1. Pipet out the medium from each well and add 400 μl Medium N(S1).
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QNS-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section using Medium N(S1) in place of Medium S to prepare
QNS mixture.
Medium Change and Drug Selection
1.

Transfer 2 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
○ If more than 90% of the cells show resistance to puromycin at the concentration used on Day 1, consider
increasing its concentration.
2. Pipet out the medium from each well and add 500 μl Medium N(S1) with puromycin.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 3< 1 hourMedium Change
IMPORTANT! It is optional, but recommended, to include the PBS wash if cell death/floating cells are observed.
1. Warm Medium N(S1) at room temperature for 20-30 minutes.
2. Transfer 2 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
3. Pipet out the old medium from each well and* add 500 µl Medium N(S1) with puromycin.
○ IMPORTANT! For medium change, a small volume of the old medium (i.e., just enough to cover thesurface of the well) should be left. The fresh medium should be applied along the wall of the well very slowly.
○ *(Optional) Slowly add 500 µl PBS alongside the wall of each well to avoid lifting attached cells. Gently
pipet out PBS before adding 500 µl Medium N(S1) with puromycin.
4. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 4

4-6 hours

Media Preparation1.

Prepare Medium N(S1P) using the volumes indicated in the table below.

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○
○

Warm Medium N(S1) and Component P at room temperature for 20-30 minutes.
Tap the Component P tube 3 times and then briefly spin it down before use.
Reagents

VolumeMedium N(S1)

8 ml

Component P

8 µl

Plate/Coverslip Preparation
IMPORTANT! Cells can be plated on glass coverslips or in wells of a 24-well plate depending on the desired application.
Wells are better suited for functional assays, whereas coverslips work best for immunostaining and imaging. If using glass
coverslips, follow all instructions starting at Step 1. If not, start at Step 1 and skip Steps 2 and 3. For first time users, werecommend only passaging two of the cultures and maintaining the other cultures (or any cultures less than 40% confluent)
without passaging. Prepare one coverslip or well for each well being passaged.
1. (Coverslip only) Soak 12-mm glass coverslips and the tips of forceps in 100% ethanol for 3 minutes.
2. (Coverslip only) One by one, air dry each coverslip for 1 minute or until completely dry and put it into a well of the
24-well plate using the sterilized forceps.
3. Vortex ornithine briefly and centrifuge it at a maximum speed for 1 minute.
4. Add 300 μl ornithine to the surface of each glass coverslip or well.
IMPORTANT! If using coverslips, ensure they are fully submerged under solutions from Step 5 onwards.
5. Incubate the plate at 37°C, 5% CO2 for at least 2 hours (or at 4°C overnight one day before plating).
6. Thaw laminin and chill 1.5 mL PBS on ice for 20-30 minutes.
7. Add 15 µl laminin to the chilled PBS. Mix well.
○ All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
8. Aspirate the supernatant from each coverslip/well and add 500 μl PBS.
9. Repeat Step 8.
10. Aspirate PBS from each coverslip/well and add 300 μl diluted laminin.
11. Incubate the plate at 37°C, 5% CO2 for at least 2 hours.
12. After the laminin incubation, pipet out most of the supernatant, but not all, from each coverslip or well.
13. Rinse with 500 μl PBS.
14. Pipet out most of the PBS, but not all, from each well.
15. Repeat Steps 13-14.
16. Add 300 μl Medium N(S1P) to each well or coverslip.
17. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care.Steps 2-10 below are critical. Perform these steps one well at a time. Refer to the images below
to successfully manage cell treatment and dissociation.

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1.
2.
3.
4.
5.
6.
7.

Make sure that Solution D1 is at room temperature for at least 1 hour before use.
Pipet out the old medium from one well and add 500 μl PBS.
Pipet out the PBS from the well and add 80 μl Solution D1.
Rock the plate 3 times to spread the Solution D1 evenly.
Incubate the culture plate at 37°C, 5% CO2 for 3 minutes.
Carefully pipet out Solution D1 from the well using a P200 pipettor and add 200 µl Medium N(S1P).
Disperse the medium quickly over the bottom surface of the well by pipetting 6-8 times to detach cells using a P200
pipettor.
8. Observe cells and/or cell aggregates floating in the well under a microscope. It is normal that 10-20% of cells
remain attached to the well bottom after pipetting. These clusters of cells are not supposed to be lifted. Do not
attempt to detach all of the cells remaining on the well bottom.
9. Gently pipet the cell suspension up and down in the well up to 5 times to break the cell aggregates using a P200
pipettor. Excessive pipetting can damage the already-suspended neuronal cells.
10. Collect 200 μl cell suspension from the well with a P200 pipettor set to 100 μl and transfer all the cells to a new wellor glass coverslip coated with ornithine and laminin 100 μl at a time.
○ IMPORTANT! If cultures are >70% confluent on Day 3, transfer only half of the cell suspension (100 μl) toeach new well or glass coverslip to avoid excessively high cell density. Add 100 μl Medium N(S1P) after cellsuspension is transferred to bring the total volume up to 200 μl. Please keep in mind that this protocol
supports the maintenance of up to 4 newly prepared wells.
○ When transferring the cell suspension to a coverslip, bring the tip very close to the coverslip and pipet
slowly to mount cells on the coverslip.
11. Repeat Steps 2-10 for the rest of the wells.
12. the culture plate at 37°C, 5% CO2 overnight.
Medium Change (Unpassaged Cultures Only)
1.

Pipet out the old medium from each unpassaged well and add 500 μl Medium N(S1P).
○ IMPORTANT! For the wells that are not passaged, the attachment of cells is weaker and so a small volumeof the old medium (i.e., just enough to cover the surface of the well) should be left. The fresh medium
should be applied along the wall of the well very slowly.
2. Incubate the culture plate at 37°C, 5% CO2 overnight.

Days 5-9< 1 hourMedium Change
IMPORTANT! It is optional, but recommended, to include the below PBS wash if cell death/floating cells are observed.
1. Warm Medium N(S1P) at room temperature for 20-30 minutes.
2. Pipet out 400 µl of the old medium from each well and* add 400 µl Medium N(S1P).
○ IMPORTANT! For the wells that are not passaged, the attachment of cells is weaker and so a small volumeof the old medium (i.e., just enough to cover the surface of the well) should be left. The fresh medium
should be applied along the wall of the well very slowly.
○ *(Optional) Slowly add 500 µl PBS alongside the wall of each well to avoid lifting attached cells. Gently
pipet out PBS before adding 800 µl Medium N(S1P).
3. Incubate the culture plate at 37°C, 5% CO2 .
4. Repeat steps 1-3 every 2-3 days.

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9Day 10
Assay or Continuous Maturation●●

Differentiated neurons can be observed on Day 5. For more mature neurons, we recommend culturing cells until
Day 10. From Day 10, users may maintain differentiated neurons in the medium best suited for their needs, though
we recommend Quick-Neuron™ Sensory Maintenance Medium, Catalog Number: SS-MM.
Differentiation into sensory neurons after using Quick-Neuron™ Sensory - mRNA Kit (Small) can be confirmed withthe markers SCN9A, PRPH, ISL1, and BRN3A.

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10Appendix

Figure 1. Representative phase contrast images of Quick-Neuron™ Sensory - mRNA Kit cell cultures on days 1, 2, 3, 4, 6, 9, and 10
post-differentiation (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Sensory - mRNA Kit cell cultures shows typical neurite growth and expressionof PRPH, TUBB3, BRN3A, and ISL1 on day 10 post-differentiation (scale bar = 100 μm). Staining conditions: Peripherin Rabbit
polyclonal antibody (ThermoFisher, Catalog number: PA110018, 1:2000 dilution) was used in combination with a secondary antibody(Invitrogen, Catalog number: A32731 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488,
1:500 dilution). Anti-β-III tubulin monoclonal antibody (R&D Systems, Catalog Number: MAB1195, 1:250 dilution) in combination with asecondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
Alexa Fluor 594, 1:500 dilution). BRN3A(14A6):mouse antibody (Santa Cruz Biotechnology, Catalog number:sc-8429 ,1:200 dilution) in

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combination with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed
Secondary Antibody, Alexa Fluor 594, 1:500 dilution). ISL1 (DSHB, Catalog number: 39.4D5, 1:200 dilution) antibody in combination
with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

Figure 3. Real-time quantitative PCR analysis of expression levels of sensory neuron-associated genes SCN9A, PRPH, ISL1, and
BRN3A were examined. The graph shows comparison of gene expression in Quick-Neuron™ - Sensory mRNA Culture on day 10 withgene expression in Human Dorsal Root Ganglion total RNA (TaKaRa, Catalog Number: 636150). The relative gene expression isnormalized to phosphoglycerate kinase 1 (PGK1), and then calculated as a fold induction relative to hiPSCs as a control. Error bars show
standard deviation. Primers used are listed in Table 1.

Table 1. List of PCR primers used in Figure 3
Gene

Forward primer

Reverse primer

Primer concentration

SCN9A

CCGTGGAACTGGCTGGATTTTTGTCTTCAGGCCTGGGATT

250 nM

PRPH

GCCTGGAACTAGAGCGCAAG

CCTCGCACGTTAGACTCTGG

250 nM

ISL1

TCTGTGGGCTGTTCACCAACTGTA

GCCGCAACCAACACATAGGGAAAT

250 nM

BRN3A

GCACACATTCACACAGTGGTAACAG

AGGCTCAGCAGACATACATGGACAG

250 nM

PGK1

GTATGCTGAGGCTGTCACTCG

CCTTCCAGGAGCTCCAAACTGG

250 nMelixirgensci.com

12