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

[STEPS]
?. [Before starting]
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 .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.
on ice
?. [Prepare lysis plates]
Prepare lysis buffer mix   ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Guanidine Hydrochloride (8000mM ; Optional)0mM - 50mM0.00--3Poly-ethylene Glycol 8000 (50% solution)5%0.40441644Triton X-100 (10% solution)0.1%0.033.312.35ERCC spike-ins (Optional)----6RNAse Inhibitor (40u/uL)0.5u/uL0.044.115.47OligodT30VN (100uM)0.5uM0.022.28.28dNTPs (25mM/each)0.5mM/each0.088.832.89Nuclease Free Water2.43267.6 997.310Total3 uL330 uL1230 uLThe 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  lysis buffer to each well of a 96/384 well plate.Quick centrifugation to collect the lysis buffer before storage until use.
ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Guanidine Hydrochloride (8000mM ; Optional)0mM - 50mM0.00--3Poly-ethylene Glycol 8000 (50% solution)5%0.40441644Triton X-100 (10% solution)0.1%0.033.312.35ERCC spike-ins (Optional)----6RNAse Inhibitor (40u/uL)0.5u/uL0.044.115.47OligodT30VN (100uM)0.5uM0.022.28.28dNTPs (25mM/each)0.5mM/each0.088.832.89Nuclease Free Water2.43267.6 997.310Total3 uL330 uL1230 uL
3 µl
?. [Sample collection]
Sort single cells into  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  freezer or dry-ice.
3 µl
-80 °C
?. [Cell lysis]
Remove the plate of sorted cells from the -80 freezer and incubate in a thermocycler with heated lid at for , followed by a  hold (keeping the storage seal sheet on the plate, unless damaged or loose).
72 °C
4 °C
?. [Reverse Transcription]
While the plate is incubating as per step 4, prepare the following Reverse transcription master-mix.  ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Tris-HCl pH 8.3 (1M)25mM0.111413NaCl (1M)30mM0.1213.249.24MgCl2 (100mM)2.5mM0.111415GTP (100mM)1mM0.044.416.46DTT (100mM)8mM0.3235.2131.27RNase Inhibitor (40u/uL)0.5u/uL0.055.520.58TSO (100uM)2uM0.088.832.89Maxima H-minus RT enzyme (200U/uL)2u/uL0.044.416.410Nuclease Free Water0.1516.561.511Total1uL110uL410uLAdd RT mix to each well of a 96/384 well plate.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;  ABC142 °C90 min1x250 °C2 min10x342 °C2 min485 °C5 min1x
ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Tris-HCl pH 8.3 (1M)25mM0.111413NaCl (1M)30mM0.1213.249.24MgCl2 (100mM)2.5mM0.111415GTP (100mM)1mM0.044.416.46DTT (100mM)8mM0.3235.2131.27RNase Inhibitor (40u/uL)0.5u/uL0.055.520.58TSO (100uM)2uM0.088.832.89Maxima H-minus RT enzyme (200U/uL)2u/uL0.044.416.410Nuclease Free Water0.1516.561.511Total1uL110uL410uL
1 µl
ABC142 °C90 min1x250 °C2 min10x342 °C2 min485 °C5 min1x
?. [Preamplification PCR]
Start preparing the PCR mix, when the incubation of the reverse transcription reaction is near completion, by combining the following components. ! 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.  ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Kapa  HiFi HotStart buffer (5X)1X2.02208203dNTPs (25mM/each)0.3mM/each0.1213.249.24MgCl2 (100mM)0.5mM0.055.520.55Fwd Primer (100uM)0.5uM0.055.520.56Rev Primer (100uM)0.1uM0.011.14.17Polymerase (1U/uL)0.02U/uL0.222828Nuclease Free Water3.57392.71463.79Total6uL660uL2460uLAdd 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.  ABCD1StepTemperatureTimeCycles2Initial denaturation98 °C3 min1x3Denaturation98 °C20 sec18-25x4Annealing65°C30 sec5Elongation72 °C4 min6Final Elongation72 °C5 min1x7Hold4 °CHold
ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Kapa  HiFi HotStart buffer (5X)1X2.02208203dNTPs (25mM/each)0.3mM/each0.1213.249.24MgCl2 (100mM)0.5mM0.055.520.55Fwd Primer (100uM)0.5uM0.055.520.56Rev Primer (100uM)0.1uM0.011.14.17Polymerase (1U/uL)0.02U/uL0.222828Nuclease Free Water3.57392.71463.79Total6uL660uL2460uL
6 µl
ABCD1StepTemperatureTimeCycles2Initial denaturation98 °C3 min1x3Denaturation98 °C20 sec18-25x4Annealing65°C30 sec5Elongation72 °C4 min6Final Elongation72 °C5 min1x7Hold4 °CHold
?. [cDNA purification (preferable but optional)]
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  AB1 ReagentAmount2PEG 800011 g
3NaCl, 5M
10 mL4Tris-HCL, 1M, pH 8.0
500 μL5EDTA, 0.5M
100 μL
6IGEPAL, 10% solution
50 μL7Sodium Azide, 10% solution
250 μL8UltraPure Waterup to 49 mL9Total 49 mLAdd all ingredients into a  falcon tube, but do not add the total amount of water until after PEG is completely solubilized.  Incubate at and vortex regularly until PEG is completely dissolved.Resuspend bead stock carefully (Sera-Mag Speed Beads).Pipette  of bead suspension into a 1.5 mL tube. Place on magnet stand  Remove supernatant.Add 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  10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads.Add to PEG solution above and mix well.
AB1 ReagentAmount2PEG 800011 g
3NaCl, 5M
10 mL4Tris-HCL, 1M, pH 8.0
500 μL5EDTA, 0.5M
100 μL
6IGEPAL, 10% solution
50 μL7Sodium Azide, 10% solution
250 μL8UltraPure Waterup to 49 mL9Total 49 mL
50 ml
40 °C
1000 µl
1000 µl
900 µl
?. [cDNA purification (preferable but optional)]
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  for .3. Place on magnet and allow beads to settle. Roughly .4. Discard supernatant, and wash once with  /  of freshly prepared 80% Ethanol for 384 / 96 well plates respectively.5. Remove Ethanol and let the beads air dry for  - 6. Elute cDNA in  UltraPure Water, resuspend beads and incubate for .
0 Room temperature
20 µl
100 µl
12 µl
?. [DNA concentration measurement and normalization (Optional, but recommended)]
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 / 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  of cDNA to each well. Incubate asssay for  at 7. Use a plate reader, to measure fluorescence (504nM Excitation/ 531nM Emission)

8. Calculate cDNA concentration. 9. Calculate water needed to dilute cDNA to.
49 µl
99 µl
1 µl
0 Room temperature
1 µl
?. [DNA concentration measurement and normalization (Optional, but recommended)]
1. Prepare normalization plate by adding the calculated water volumes to each well.2. Add  of preamplified cDNA to each well.
1 µl
?. [Quality Control check (Optional)]
Check the cDNA preamplification library content and quality on a Agilent Bioanalzyer, using High Sensitivity DNA Analysis chips.
?. [Tagmentation]
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.  ABCD1 ReagentAmount (uL)Concentration in 4X2Tris-HCl pH 7.5 (1M)4040mM3MgCl2 (100mM)20020mM4Dimethylformamide (DMF)20020%5UltraPure Water5606Total1000 uL2. 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..   ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Tagmentation buffer (4x)1X0.5552053Amplicon Tagmentation Mix (Tn5)0.088.832.84UltraPure water0.4246.2172.25Total1uL110uL410uL3. Dispense  of Tagmentation mix to a new 96 or 384 well plate.4. Add  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  for .6. To strip off the Tn5 from the DNA, add  of 0.2% SDS to each well. Centrifuge quickly and incubate for .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  Nextera Index primers to each well as follows  ABCDE1ReagentReaction conc.uL per. reaction2Custom S50X index  primer (0.5uM)0.1uM0.753Custom  N70X index primer (0.5uM)0.1uM0.759. Prepare Tagmentation PCR mix.  ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Phusion  HF buffer (5X)1X1.41545743dNTPs (25mM/each)0.2mM/each0.066.2234Phusion HF (2U/uL)0.01U/uL0.043.914.45H2O1.51166618.76Total3uL330uL1230uL 10. Add  of Tagmentation PCR mix to each well, centrifuge quickly and incubate in a thermocycler using the following PCR program.  ABCD1StepTemperatureTimeCycles2Gap-filling72 °C3 min1x3Initial denaturation98 °C3 min1x4Denaturation98 °C10 sec12x5Annealing55 °C30 sec6Elongation72 °C30 sec7Final Elongation72 °C5 min1x8Hold4 °CHold
Dimethylformamide (DMF) should be handled in a fume hood and according to local safety regulations.
ABCD1 ReagentAmount (uL)Concentration in 4X2Tris-HCl pH 7.5 (1M)4040mM3MgCl2 (100mM)20020mM4Dimethylformamide (DMF)20020%5UltraPure Water5606Total1000 uL
ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Tagmentation buffer (4x)1X0.5552053Amplicon Tagmentation Mix (Tn5)0.088.832.84UltraPure water0.4246.2172.25Total1uL110uL410uL
1 µl
1 µl
55 °C
0.5 µl
1.5 µl
ABCDE1ReagentReaction conc.uL per. reaction2Custom S50X index  primer (0.5uM)0.1uM0.753Custom  N70X index primer (0.5uM)0.1uM0.75
ABCDE1ReagentReaction conc.uL per. reaction96 well plate384 well plate2Phusion  HF buffer (5X)1X1.41545743dNTPs (25mM/each)0.2mM/each0.066.2234Phusion HF (2U/uL)0.01U/uL0.043.914.45H2O1.51166618.76Total3uL330uL1230uL
3 µl
ABCD1StepTemperatureTimeCycles2Gap-filling72 °C3 min1x3Initial denaturation98 °C3 min1x4Denaturation98 °C10 sec12x5Annealing55 °C30 sec6Elongation72 °C30 sec7Final Elongation72 °C5 min1x8Hold4 °CHold
?. [Library clean-up]
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  at 2. Place on magnet and allow beads to settle. Roughly .4. Discard supernatant, and wash twice with >=  freshly prepared 80% Ethanol.5. Remove Ethanol and let the beads air dry for at least 6. Elute cDNA in  UltraPure Water, resuspend beads and incubate for .
55 Room temperature
1000 µl
40 µl
?. [(Optional) Size selection via Gel-cutting and extraction]
To further select for longer tagmented fragments, an optional step including a size selection step can be included.1. Load  of the eluted tagmented library from step 13 into a 2% Agarose E-Gel EX together with 50bp DNA ladder.2. Run gel for 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.
20 µl
?. [Final Library Quantification]
Run the final library on a Agilent Bioanalyzer (High Sensitivity DNA), to inspect the quality and median base-pair length of your library.Use Qubit fluorometer or similar to quantify the library.Calcutate the final library concentration, using above metrics.
?. [Sequencing]
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 sequncers 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". Empircal investigation or a pilot run is always adviced, if possible.
?. [Primary Data processing]
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).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-R denotes the runfolder and you may redirect the fastq output to a different folder with the -o option.
?. [Primary Data processing]
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.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:Be sure to use full paths to all files and folders. For further descriptions of the individual options visit the zUMIs GitHub repository wikiNow, simply start zUMIs with the following command: