Feb 13, 2020

Version 2

<figure><img src="image_1.png" /></figure>

# Smart-seq3 Protocol V.2

DOI

https://dx.doi.org/10.17504/protocols.io.bbpyimpw

<figure><img src="image_2.png" /></figure>

Michael Hagemann-Jensen¹, Christoph Ziegenhain¹, Ping Chen¹, Daniel Ramsköld¹, Gert-Jan Hendriks¹, Anton J.M Larsson¹, Omid R. Faridani¹, Rickard Sandberg¹

¹Karolinska Institute Stockholm

<figure><img src="image_3.png" /><figcaption>Michael Hagemann-Jensen</figcaption></figure>

Karolinska Institutet

Create & collaborate more with a free account

Edit and publish protocols, collaborate in communities, share insights through comments, and track progress with run records.

Create free account

OPEN

<figure><img src="image_4.png" /></figure>

ACCESS

<figure><img src="image_5.png" /></figure>

DOI: https://dx.doi.org/10.17504/protocols.io.bbpyimpw

**Protocol Citation:** Michael Hagemann-Jensen, Christoph Ziegenhain, Ping Chen, Daniel Ramsköld, Gert-Jan Hendriks, Anton J.M Larsson, Omid R. Faridani, Rickard Sandberg 2020. Smart-seq3 Protocol. protocols.io

https://dx.doi.org/10.17504/protocols.io.bbpyimpw
<figure><img src="image_6.png" /></figure>

**License:** This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

**Created:** January 23, 2020

**Last Modified:** February 13, 2020

**Protocol Integer ID:** 32216

**Keywords:** protocol
<figure><img src="image_7.png" /></figure>

## Guidelines

* If you are pro, and want to change the volumes indicated in the protocol, make sure that the reaction concentrations stay similar. Also ensure that the spillover concentrations into the next reaction also stay similar. Failing to do so can lead to decreased performance of the protocol. In terms of the tagmentation reaction make sure to keep the ratio of cDNA input to Nextera TN5 amount, if you contemplate to miniaturize this reaction.

* The current protocols is based on the TN5 from Nextera Xt kit. However Illumina TDE1, works as well.

* For determining PCR cycles, a good general guideline is to add 1-2 cycles more than using Smart-seq2. However as always, this is good to empirically test first, before running important samples.

### List of oligos:

<table><thead><tr><td>Oligo</td><td>Vend or</td><td>Purificati on</td><td>Working concentrati on</td><td>Sequence</td></tr></thead><tbody><tr><td>Smartseq3_Oligo dT30VN</td><td>IDT</td><td>HPLC</td><td>100uM</td><td>/5Biosg/ACGAGCATCAGCAGCATACGATTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTVN</td></tr><tr><td>Smartseq3_N8_T SO</td><td>IDT</td><td>RNaSe- Free HPLC</td><td>100uM</td><td>/5Biosg/AGAGACAGATTGCGCAATGNNNNN NNNrGrGrG</td></tr><tr><td>Fwd_PCR_primer</td><td>IDT</td><td>HPLC</td><td>100uM</td><td>TCGTCGGCAGCGTCAGATGTGTATAAGAGAC AGATTGCGCAA*T*G</td></tr><tr><td>Rev_PCR_primer</td><td>IDT</td><td>HPLC</td><td>100uM</td><td>ACGAGCATCAGCAGCATAC*G*A</td></tr></tbody></table>

* phosphorothioate bonds

* It is absolute fine to use standard desalting instead of HPLC, both works fine in our hands, same goes for hand-mixed vs machine mixed degenerate bases. Using the regular DNA oligos service at IDT should provide based on their QC full length oligos.

* We use custom Nextera Indexes primers (standard 25 nmol oligo preps from IDT, delivered at 200 nM concentration in IDTE buffer) and we typically get performance that is indistinguishable from Illumina's primers.

For making your own primers, we recommend using the "DNABarcodes" R package. using the following settings:

Barcode length: 10 bp (or 8bp like Illumina primers, depending on the amount of cells you need indexed and sequenced at the same time)

Minimal levenshtein distance: 3

Filter out homopolymers >= 3

Filter for uneven GC content
<figure><img src="image_8.png" /></figure>

Additionally, there seems to be an artifact on the NovaSeq platform for i5 index primers starting with the bases "AC", so we recommend to avoid those too!

(see supplementary information in this paper: https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4703-0#Sec13)

* For troubleshooting: feel free to leave comments or message directly.
<figure><img src="image_9.png" /></figure>

# Materials

## MATERIALS

Triton X-100 Merck MilliporeSigma (Sigma-Aldrich) Catalog #T8787-50ML

Agilent High Sensitivity DNA Kit Agilent Technologies Catalog #5067-4626

Nextera XT DNA Library Preparation Kit Illumina, Inc. Catalog #FC-131-1096

QIAquick Gel Extraction Kit Qiagen Catalog #28704

DNA LoBind Tubes, 1.5 mL Eppendorf Catalog #0030108051

Recombinant RNAse Inhibitor Takara Bio Inc. Catalog #2313A

Dithiothreitol (DTT) Thermo Fisher Scientific Catalog #707265ML

dNTP Set 100 mM Solutions Thermo Fisher Scientific Catalog #R0182

UltraPure&trade; DNase/RNase-Free Distilled Water Thermo Fisher Catalog #10977035

EDTA (0.5 M), pH 8.0, RNase-free Thermo Fisher Catalog #AM9260G

SDS, 10% Solution, RNase-free Thermo Fisher Catalog #AM9822

Maxima H Minus Reverse Transcriptase (200 U/&micro;L) Thermo Fisher Catalog #EP0751

Poly Ethylene Glycol (PEG) 8000 Merck MilliporeSigma (Sigma-Aldrich) Catalog #89510-250G-F

Sodium Chloride (5M) Invitrogen - Thermo Fisher Catalog #AM9760G

Magnesium Chloride (1M Solution) Invitrogen - Thermo Fisher Catalog #AM9530G

GTP (Tris buffered solution 100mM) Thermo Scientific Catalog #R1461

Trizma-base Merck MilliporeSigma (Sigma-Aldrich) Catalog #T6791-100G

KAPA HiFi Hotstart PCR kit Roche Catalog #KK2502

Phusion High-Fidelity DNA Polymerase (2 U/µL) Thermo Scientific Catalog # F530L

Sera-Mag Speed Beads GE Healthcare Catalog #65152105050250

Sodium Azide Merck MilliporeSigma (Sigma-Aldrich) Catalog #S2002-100G

IGEPAL® CA-630 Merck MilliporeSigma (Sigma-Aldrich) Catalog #I8896

Armadillo PCR Plate 96-well clear semi-skirted white wells Thermo Scientific Catalog #AB3596

Armadillo PCR Plate 384-well Thermo Scientific Catalog # AB2384B

QuantiFluor® dsDNA System Promega Catalog #E2670

E-Gel™ EX Agarose Gels 2% Thermo Scientific Catalog #G402002

2-propanol Merck MilliporeSigma (Sigma-Aldrich) Catalog #I9516

NN-Dimethylformamide Merck MilliporeSigma (Sigma-Aldrich) Catalog #D4551
<figure><img src="image_10.png" /></figure>

Troubleshooting
<figure><img src="image_11.png" /></figure>

## Before starting

1. This protocol should be carried out in a clean environment. Use ethanol, RNAseZAP, DNA-OFF, or similar to prepare work bench before start.

Work quickly and preferably on ☀ On ice.

Prepare master-mixes right before use.

Use multichannel pipettes, liquid dispensers etc. to dispense the master-mixes. Avoid pipetting up and down, to minimize the loss of material.

* Take a look at the Guidelines section for more info about Oligos etc. used in this protocol.

## Prepare lysis plates

2. Prepare **lysis buffer mix**

<table><thead><tr><th>Reagent</th><th>Reaction conc.</th><th>uL per. reaction</th><th>96 well plate</th><th>384 well plate</th></tr></thead><tbody><tr><td>Guanidine Hydrochloride (8000mM; Optional)</td><td>0mM - 50mM</td><td>0.00</td><td>-</td><td>-</td></tr><tr><td>Poly-ethylene Glycol 8000 (50% solution)</td><td>5%</td><td>0.40</td><td>44</td><td>164</td></tr><tr><td>Triton X-100 (10% solution)</td><td>0.1%</td><td>0.03</td><td>3.3</td><td>12.3</td></tr><tr><td>ERCC spike-ins (Optional)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>RNAse Inhibitor (40u/uL)</td><td>0.5u/uL</td><td>0.04</td><td>4.1</td><td>15.4</td></tr><tr><td>OligodT30VN (100uM)</td><td>0.5uM</td><td>0.02</td><td>2.2</td><td>8.2</td></tr><tr><td>dNTPs (25mM/each)</td><td>0.5mM/each</td><td>0.08</td><td>8.8</td><td>32.8</td></tr><tr><td>Nuclease Free Water</td><td></td><td>2.43</td><td>267.6</td><td>997.3</td></tr><tr><td>Total</td><td></td><td>3 uL</td><td>330 uL</td><td>1230 uL</td></tr></tbody></table>

Reaction concentrations for PEG8000, OligodT30VN and dNTPs, are adjusted to and reflect their concentration in the reverse transcription reaction (4uL)
<figure><img src="image_12.png" /></figure>

The lysis master-mix contains PEG! Ensure that PEG is fully mixed into solution, by either pipetting up and down until the liquid is clear, or start with vortexing the required master-mix volume of water and PEG together before adding the remaining reagents.

Add ⬇ 3 µL lysis buffer to each well of a 96/384 well plate.

Quick centrifugation to collect the lysis buffer before storage until use.

## Sample collection

3. Sort single cells into ⬇ 3 µL lysis in either 96 or 384 wells.

Seal with appropriate seals (-80 C to >100 C) and centrifuge the finished sorted plate immediately after, and transfer it to a ⬇ -80 °C freezer or dry-ice.

## Cell lysis

4. Remove the plate of sorted cells from the -80 freezer and incubate in a thermocycler with heated lid at ⬇ 72 °C for 🕒 00:10:00, followed by a ⬇ 4 °C hold (keeping the storage seal sheet on the plate, unless damaged or loose).

## Reverse Transcription

5. While the plate is incubating as per **step 4**, prepare the following **Reverse transcription master-mix**.

<table><thead><tr><th>Reagent</th><th>Reaction con c.</th><th>uL per. reac tion</th><th>96 well plat e</th><th>384 well plat e</th></tr></thead><tbody><tr><td>Tris-HCl pH 8.3 (1M)</td><td>25m M</td><td>0.1</td><td>11</td><td>41</td></tr><tr><td>NaCl (1M)</td><td>30m M</td><td>0.12</td><td>13.2</td><td>49.2</td></tr></tbody></table>
<figure><img src="image_13.png" /></figure>

<table><tr><td>MgCl<sub>2</sub> (100 mM)</td><td>2.5mM</td><td>0.1</td><td>11</td><td>41</td></tr><tr><td>GTP (100 mM)</td><td>1mM</td><td>0.04</td><td>4.4</td><td>16.4</td></tr><tr><td>DTT (100 mM)</td><td>8mM</td><td>0.32</td><td>35.2</td><td>131.2</td></tr><tr><td>RNa se Inhibitor (40u /uL)</td><td>0.5u /uL</td><td>0.05</td><td>5.5</td><td>20.5</td></tr><tr><td>TSO (100 uM)</td><td>2uM</td><td>0.08</td><td>8.8</td><td>32.8</td></tr><tr><td>Maxi ma H-minu s RT enzy me (200 U/uL )</td><td>2u/u L</td><td>0.04</td><td>4.4</td><td>16.4</td></tr><tr><td>Nucl ease Free Wat er</td><td></td><td>0.15</td><td>16.5</td><td>61.5</td></tr><tr><td>Tota l</td><td></td><td>1uL</td><td>110u L</td><td>410 uL</td></tr></table>

Add 1 µL RT mix to each well of a 96/384 well plate.

<figure><img src="image_14.png" /></figure>

Replace the storage seal with a PCR seal. Ensure that the plate is properly sealed, to avoid evaporation.

Do a quick centrifugation to collect reaction at the bottom, before incubating the plate in a thermocycler at;

<table><tr><td>42 °C</td><td>90 min</td><td>1x</td></tr></table>
<table><tr><td>50 ° C</td><td>2 min</td><td rowspan="2">10x</td></tr><tr><td>42 ° C</td><td>2 min</td></tr><tr><td>85 ° C</td><td>5 min</td><td>1x</td></tr></table>

## Preamplification PCR

6

Start preparing the **PCR mix**, when the incubation of the reverse transcription reaction is near completion, by combining the following components.

¹ Note that the KAPA DNA polymerase has a 3-5' exonuclease activity that is not HotStart. Therefore add polymerase just before using the master-mix.

<table><thead><tr><th>Reagent</th><th>Reaction con c.</th><th>uL per. reac tion</th><th>96 well plat e</th><th>384 well plat e</th></tr></thead><tbody><tr><td>Kapa HiFi HotStart buffer (5X)</td><td>1X</td><td>2.0</td><td>220</td><td>820</td></tr><tr><td>dNTPs (25mM/each)</td><td>0.3 mM/ each</td><td>0.12</td><td>13.2</td><td>49.2</td></tr><tr><td>MgCl2 (100mM)</td><td>0.5m M</td><td>0.05</td><td>5.5</td><td>20.5</td></tr><tr><td>Fwd Primer (100uM)</td><td>0.1u M</td><td>0.01</td><td>1.1</td><td>4.1</td></tr><tr><td>Rev Primer (100uM)</td><td>0.1u M</td><td>0.01</td><td>1.1</td><td>4.1</td></tr><tr><td>Polymerase (1U/uL)</td><td>0.02 U/uL</td><td>0.2</td><td>22</td><td>82</td></tr><tr><td>Nuclease Free Water</td><td></td><td>3.61</td><td>379.1</td><td>1480.1</td></tr><tr><td><strong>Total</strong></td><td></td><td><strong>6uL</strong></td><td><strong>660 uL</strong></td><td><strong>246 OuL</strong></td></tr></tbody></table>

Add 6 µL PCR mix to each well of a 96/384 well plate.
Quick centrifugation to collect reaction at the bottom, before running the following PCR program in a thermocycler.

<table><thead><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Cycles</th></tr></thead><tbody><tr><td>Initiation</td><td>98°C</td><td>3 min</td><td>1x</td></tr><tr><td>Denaturation</td><td>98°C</td><td>20 sec</td><td rowspan="3">18-25x</td></tr><tr><td>Annaling</td><td>65°C</td><td>30 sec</td></tr><tr><td>Elongation</td><td>72°C</td><td>4 min</td></tr><tr><td>Final Elongation</td><td>72°C</td><td>5 min</td><td>1x</td></tr><tr><td>Hold</td><td>4°C</td><td>Hold</td><td></td></tr></tbody></table>

The PCR cycle number depends on the input, and is very cell-type specific. See the Guidelines & warnings for help determining PCR cycles needed.

## cDNA purification (preferable but optional)

7. Before purification prepare **22% PEG Clean-up Beads** used for cleaning up the preamplified cDNA. These beads perform similar to Ampure XP beads. Beads are prepared as per mcSCRB-seq protocol
<figure><img src="image_15.png" /></figure>

Citation

Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard (2026). mcSCRB-seq protocol. Nature Communications.

dx.doi.org/10.17504/protocols.io.p9kdr4w

LINK

<table><thead><tr><td>Reagent</td><td>Amount</td></tr></thead><tbody><tr><td>PEG 800 0</td><td>11 g</td></tr><tr><td>NaCl, 5M</td><td>10 mL</td></tr><tr><td>Tris-HCL, 1M, pH 8.0</td><td>500 μL</td></tr><tr><td>EDT A, 0.5 M</td><td>100 μL</td></tr><tr><td>IGEP AL, 10% solution</td><td>50 μL</td></tr><tr><td>Sodium Azide, 10% solution</td><td>250 μL</td></tr><tr><td>Ultra Pure Water</td><td>up to 49 mL</td></tr><tr><td>Total</td><td>49 mL</td></tr></tbody></table>
<figure><img src="image_16.png" /></figure>

Add all ingredients into a ⌀ 50 mL falcon tube, but do not add the total amount of water until after PEG is completely solubilized.

Incubate at ⌀ 40 °C and **vortex regularly until PEG is completely dissolved**.

Resuspend bead stock carefully (Sera-Mag Speed Beads).

Pipette ⌀ 1000 µL of bead suspension into a 1.5 mL tube.

Place on magnet stand Remove supernatant.

Add ⌀ 1000 µL 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads. Place on magnet stand.

Remove supernatant. Repeat wash one more time.

Add ⌀ 900 µL 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads.

Add to PEG solution above and mix well.

8

1. To purify cDNA add **0.6:1** ratio of 22% PEG beads to sample, and mix by gently pipetting up and down.

2. Incubate at ⌀ Room temperature for 🕒 00:08:00.

<figure><img src="image_17.png" /></figure>

3. Place on magnet and allow beads to settle. Roughly 🕒 00:05:00.

<figure><img src="image_18.png" /></figure>

4. Discard supernatant, and wash once with ⌀ 20 µL / ⌀ 100 µL of **freshly prepared 80% Ethanol** for 384 / 96 well plates respectively.

<figure><img src="image_19.png" /></figure>

5. Remove Ethanol and let the beads air dry for 🕒 00:02:00 - 🕒 00:05:00.

<figure><img src="image_20.png" /></figure>

6. Elute cDNA in ⌀ 12 µL UltraPure Water, resuspend beads and incubate for 🕒 00:05:00.

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

## DNA concentration measurement and normalization (Optional, but recommended)
<figure><img src="image_23.png" /></figure>

9. 1. Prepare 1X TE buffer by either diluting the 20X TE buffer from the QuantiFluor® dsDNA kit or by preparing a solution of 10mM Tris-HCl, 0.1mM EDTA, pH 8.

2. Dilute the QuantiFluor® dsDNA Dye 1:400 in 1X TE buffer and mix.

3. Prepare dsDNA standards for plate read-out, according to manufacturers protocol.

4. Dispense ⬇ 49 µL / ⬇ 99 µL per well of the ready Quantiflour dye mix into black, flat-bottom 384/96 well plates, respectively.

5. Add Standards to a separate plate.

6. Add ⬇ 1 µL of cDNA to each well. Incubate asssay for 🕒 00:05:00 at 🌡 Room temperature

7. Use a plate reader, to measure fluorescence (504nM Excitation/ 531nM Emission)

8. Calculate cDNA concentration.

9. Calculate water needed to dilute ⬇ 1 µL cDNA to [M] 100 pg/uL .

10. 1. Prepare **normalization plate** by adding the calculated water volumes to each well.

2. Add ⬇ 1 µL of preamplified cDNA to each well.

Quality Control check (Optional)

11. Check the cDNA preamplification library content and quality on a Agilent Bioanalyzer, using High Sensitivity DNA Analysis chips.
## Expected result

<figure><img src="image_24.png" /></figure>

Example of one HEK cell.

## Tagmentation

12

1. Prepare 4x Tagmentation buffer as following. Aliquots of 4xTD buffer can be stored for later use. The TD buffer (2x) from Nextera Kits can also be used, however with the current small amount of ATM used, the Illumina TD buffer will at some point run out.

## Safety information

Dimethylformamide (DMF) should be handled in a fume hood and according to local safety regulations.

<table><thead><tr><td>Reagent</td><td>Amount (uL)</td><td>Concentration in 4X</td></tr></thead><tbody><tr><td>Tris-HCl pH 7.5 (1M)</td><td>40</td><td>40mM</td></tr></tbody></table>
<figure><img src="image_25.png" /></figure>

<table><tr><td>MgCl2 (100 mM)</td><td>200</td><td>20mM</td><td></td></tr><tr><td>Dimethylformamide (DMF)</td><td>200</td><td>20%</td><td></td></tr><tr><td>Ultra Pure Water</td><td>560</td><td></td><td></td></tr><tr><td>Total</td><td>1000 uL</td><td></td><td></td></tr></table>

## 2. Prepare Tagmentation mix.

* Please note that the ATM amount is a suggested starting point for 100pg/uL input, and some optimization might be necessary to reach a desired UMI-read to Internal-read ratio, based on input and celltype..

<table><thead><tr><th>Reagent</th><th>Reaction con c.</th><th>uL per. reaction</th><th>96 well plate</th><th>384 well plate</th></tr></thead><tbody><tr><td>Tagmentation buffer (4x)</td><td>1X</td><td>0.5</td><td>55</td><td>205</td></tr><tr><td>Amplicon Tagmentation Mix (Tn5)</td><td></td><td>0.08</td><td>8.8</td><td>32.8</td></tr><tr><td>Ultra Pure water</td><td></td><td>0.42</td><td>46.2</td><td>172.2</td></tr></tbody></table>
<table><tr><td>Tota</td><td></td><td>1uL</td><td>110u</td><td>410 uL</td></tr></table>

3. Dispense ⬇ 1 µL of Tagmentation mix to a new 96 or 384 well plate.

4. Add ⬇ 1 µL of **normalized 100pg/uL cDNA (step 10)** to the plate containing tagmentation mix.

5. Apply a quick spin-down of the plate before incubation in a thermocycler at ⬇ 55 °C for 🕒 00:10:00.

6. To strip off the Tn5 from the DNA, add ⬇ 0.5 µL of 0.2% SDS to each well. Centrifuge quickly and incubate for 🕒 00:05:00.

7. **Concerning Nextera Index primers: We highly suggest to design or order custom Nextera Index primers. This ensures higher flexibility while also being much cheaper in the long run! The following protocol is designed as such. If using Nextera index primers purchased from Illumina, dilute all primers 5x with UltraPure water, and proceed to use similar volume as follows.**

8. Add ⬇ 1.5 µL **Nextera Index primers** to each well as follows

<table><thead><tr><th>Reagent</th><th>Reaction con c.</th><th>uL per. reac tion</th></tr></thead><tbody><tr><td>Cust om S50 X inde x prim er (0.5 uM)</td><td>0.1u M</td><td>0.75</td></tr></tbody></table>
<table><tr><td>Cust om N70 X inde x prim er (0.5 uM)</td><td>0.1u M</td><td>0.75</td><td></td></tr></table>

9. Prepare Tagmentation PCR mix.

<table><thead><tr><th>Reagent</th><th>Reaction con c.</th><th>uL per. reac tion</th><th>96 well plat e</th><th>384 well plat e</th></tr></thead><tbody><tr><td>Phusion HF buff er (5X)</td><td>1X</td><td>1.4</td><td>154</td><td>574</td></tr><tr><td>dNT Ps (25 mM/ each )</td><td>0.2 mM/ each</td><td>0.06</td><td>6.2</td><td>23</td></tr><tr><td>Phusion HF (2U/ uL)</td><td>0.01 U/uL</td><td>0.04</td><td>3.9</td><td>14.4</td></tr><tr><td>H2O</td><td></td><td>1.51</td><td>166</td><td>618.7</td></tr><tr><td>Total</td><td></td><td>3uL</td><td>330 uL</td><td>123 0uL</td></tr></tbody></table>

10. Add 3 µL of Tagmentation PCR mix to each well, centrifuge quickly and incubate in a thermocycler using the following PCR program.

<table><thead><tr><th>Step</th><th>Temp era ture</th><th>Time</th><th>Cycl es</th></tr></thead></table>
<table><tr><td>Gap - filling</td><td>72 °C</td><td>3 min</td><td>1x</td></tr><tr><td>Initial denaturation</td><td>98 °C</td><td>3 min</td><td>1x</td></tr><tr><td>Denaturation</td><td>98 °C</td><td>10 sec</td><td rowspan="3">12x</td></tr><tr><td>Annaling</td><td>55 °C</td><td>30 sec</td></tr><tr><td>Elongation</td><td>72 °C</td><td>30 sec</td></tr><tr><td>Final Elongation</td><td>72 °C</td><td>5 min</td><td>1x</td></tr><tr><td>Hold</td><td>4 °C</td><td>Hold</td><td></td></tr></table>

## Library clean-up

13. For the final library clean-up, pool all the Tagmented cDNA (step 12) sample wells in a 1.5mL or 5mL eppendorf tube.

1. Add **0.6:1 22% PEG beads to final volume of the pooled tagmentation cDNA.** Mix gently by pipetting and incubate for 00:08:00 at **Room temperature**

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

2. Place on magnet and allow beads to settle. Roughly 00:05:00.

<figure><img src="image_28.png" /></figure>

4. Discard supernatant, and wash twice with **> = 1000 µL freshly prepared 80% Ethanol.**

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

5. Remove Ethanol and let the beads air dry for at least 00:05:00.

<figure><img src="image_31.png" /></figure>
<figure><img src="image_32.png" /></figure>

6. Elute cDNA in ⬇ 40 µL UltraPure Water, resuspend beads and incubate for

<figure><img src="image_33.png" /></figure>

00:05:00

.

(Optional) Size selection via Gel-cutting and extraction

14 To further select for longer tagmented fragments, an optional step including a size selection step can be included.

1. Load ⬇ 20 µL of the eluted tagmented library from step 13 into a **2% Agarose E-Gel EX** together with 50bp DNA ladder.

2. Run gel for 🕒 00:12:00

<figure><img src="image_34.png" /></figure>

3. After finished run, open the gel casing and cut the gel between **550bp - 2kb** using a clean scalpel or blade.

4. Purify the excised gel slice using Qiagen QIAquick Gel extraction kit according to manufacturers protocol.

Final Library Quantification

15

Run the final library on a Agilent Bioanalyzer (High Sensitivity DNA), to inspect the quality and median base-pair length of your library.
Expected result

<figure><img src="image_35.png" /></figure>

Suggestive example of a finished HEK library. A bit on the large size. Can still be sequenced.

Use Qubit fluorometer or similar to quantify the library.

Calculate the final library concentration, using above metrics.

Sequencing

16 The sequencing ready library should be sequenced on any Illumina compatible sequencer, either Single-end or Paired-end, depending on the question and need.

For final library whether gel cut/size selected or not, the expected median base-pair should be around or above 1kb. In our experience NovaSeq/HiSeq sequencers are more tolerant towards wider or longer size fragment distributions, than the NextSeq. Because of this consider increasing the loading concentration a bit to ensure proper cluster density. However "your milage may vary". Empirical investigation or a pilot run is always advised, if possible.

Primary Data processing
<figure><img src="image_36.png" /></figure>

17 After sequencing has completed successfully, binary base-call files (BCL) need to be converted to fastq.

For this, bcl2fastq should be used in the latest version (bcl2fastq v2.20).

## Software

bcl2fastq

NAME

Illumina

DEVELOPER

https://emea/support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html

SOURCE
LINK

At this stage, demultiplexing into per-cell fastq files is not necessary - a sample sheet is thus not needed.

Be sure to adjust the base mask to represent your sequencing layout and the length of your barcode reads.

Remove the option --no-lane-splitting if the same cell barcodes have been reused for different libraries on different lanes of the flow cell.

You may restrict the number of cores used with the following options:

- --loading-threads

- --processing-threads

- --writing-threads

## Command

bcl2fastq: 2×150bp dual-index

bcl2fastq: 2×150bp dual-index

bcl2fastq --use-bases-mask Y150N,I8,I8,Y150N --no-lane-splitting --create-fastq-for-index-reads -R /mnt/storage1/NextSeqNAS/191011_NB502120_0154_AHVG7JBGXB

-R denotes the runfolder and you may redirect the fastq output to a different folder with the -o option.
<figure><img src="image_37.png" /></figure>

18 After generating fastq files, the zUMIs pipeline should be used to process Smart-seq3 data to ensure correct handling of UMI reads and internal reads.

Citation

Parekh S, Ziegenhain C, Vieth B, Enard W, Hellmann I (2018)

. zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs.
GigaScience.

https://doi.org/10.1093/gigascience/giy059

LINK

Software

zUMIs

Linux

https://github.com/sdparekh/zUMIs

https://www.biorxiv.org/content/early/2017/10/18/153940

NAME

OS

REPOSITORY

SOURCE LINK

We recommend the newest version v2.5.6 at the time of this protocol.

All options are set in a configuration file following the YAML format. Here is a best
practice example:
<figure><img src="image_38.png" /></figure>

## Command

### Smartseq3.yaml

### Smartseq3.yaml

```yaml
project: Smartseq3
sequence_files:
  file1:
    name: /smartseq3/fastq/Undetermined_S0_R1_001.fastq.gz
    base_definition:
      - cDNA(23-150)
      - UMI(12-19)
    find_pattern: ATTGCGCAATG
  file2:
    name: /smartseq3/fastq/Undetermined_S0_R2_001.fastq.gz
    base_definition:
      - cDNA(1-150)
  file3:
    name: /smartseq3/fastq/Undetermined_S0_I1_001.fastq.gz
    base_definition:
      - BC(1-8)
  file4:
    name: /smartseq3/fastq/Undetermined_S0_I2_001.fastq.gz
    base_definition:
      - BC(1-8)
reference:
  STAR_index: /resources/genomes/Mouse/STAR5idx_noGTF/
  GTF_file: /resources/genomes/Mouse/Mus_musculus.GRCm38.91.gtf
  additional_STAR_params: '--clip3pAdapterSeq CTGTCTCTTATACACATCT'
  additional_files:
    - /resources/genomes/spikes/ERCC92.fa
out_dir: /smartseq3/zUMIs/
num_threads: 20
mem_limit: 50
filter_cutoffs:
  BC_filter:
    num_bases: 3
    phred: 20
  UMI_filter:
    num_bases: 2
    phred: 20
barcodes:
  barcode_num: ~
```
<figure><img src="image_39.png" /></figure>

```
barcode_file: /smartseq3/expected_barcodes.txt
automatic: no
BarcodeBinning: 1
nReadsperCell: 100
demultiplex: no
counting_opts:
introns: yes
downsampling: '0'
strand: 0
Ham_Dist: 1
write_ham: no
velocityto: no
primaryHit: yes
twoPass: no
make_stats: yes
which_Stage: Filtering
samtools_exec: samtools
pigz_exec: pigz
STAR_exec: STAR
Rscript_exec: Rscript
```

Be sure to use full paths to all files and folders. For further descriptions of the individual options visit the [zUMIs GitHub repository wiki](https://github.com/zUMIs/zUMIs/wiki)

Now, simply start zUMIs with the following command:

Command

Invoke zUMIs

Invoke zUMIs

zUMIs-master.sh -y Smartseq3.yaml
<figure><img src="image_40.png" /></figure>

## Citations

### Step 18

Parekh S, Ziegenhain C, Vieth B, Enard W, Hellmann I. zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs.

https://doi.org/10.1093/gigascience/giy059

### Step 7

Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard. mcSCRB-seq protocol

dx.doi.org/10.17504/protocols.io.p9kdr4w