TITLE: In-house automated Smart-Seq2
AUTHORS: Lira Mamanova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes the procedure for the in-house automated Smart-Seq2 workflow with off-the-shelf reagents. This set up utilises the benchtop robots and significantly reduces the experimental cost, hands-on time, as well as increase the reproducibility and workflow efficiency, while delivering a throughput of thousands of cells per day.</div><div class = "text-block">Following library construction, samples are pooled in equivolume and quantified, prior to sequencing on the Illumina  platform.</div></div>

[STEPS]
?. [Preparation of lysis buffer plates and single-cell samples]
For each well, prepare the Triton buffer by adding  of RNAse inhibitor and  of diluted (1/500,000) ERCCs to the  of  Triton X-100. Prepare an RLT lysis buffer by using  of RLT buffer (Qiagen);  of DTT (2M) and  of diluted (1/500,000) ERCCs. Dispense  of Triton lysis buffer or  of RLT lysis buffer into each well of a 96-well plate.
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A single-cell contains a very low amount of RNA. Therefore, extra effort should be taken to avoid sample degradation and reagent contamination. All work surfaces and equipment should be decontaminated with DNA and RNAZap before and after the experiment. RNA related work should be carried out in a specially designated cleanroom or in a laminar flow hood equipped with UV light for sterilization. When working with a few batches of samples, we would advise pre-aliquotting reagents, especially if the reagent supplied in a large volume.For reagent dispensing steps, we use the Formulatrix Mantis microfluidic liquid handler, and for all purification steps, we use a Zephyr automated liquid-handling robot.Steps 1-12 should be performed in specially designated RNAse free areas, keeping reagents and cells chilled at all times.The Formulatrix Mantis microfluidic liquid handler is positioned in a laminar flow hood. The lysis buffer dispensing step can be performed on alternative liquid handling platforms or manually. The reagents were dispensed from a 0.2 ml or 1 ml tip inserted into the High Volume (HV) Silicone based Mantis chip. We recommend having an individual set of chips for various reagent dispensing. Clean the dispensing chips prior to use by dispensing 50-100 μl of solutions into the waste plate in the following order: Water => 10% Bleach (incubate for  ) => Water=> 80% EtOH=>Water. Repeat these procedures after finishing the reagent dispensing.
0.2 µl
0.02 µl
1.8 µl
9.8 µl
0.2 µl
0.02 µl
2 µl
10 µl
We recommend to seal dispensed plates and centrifuge them immediately (  for   at  ) to spin down the lysis buffer to the bottom of the well to prevent a sample degradation.Sealed Lysis buffer plates can be stored at   prior to cell sorting for
?. [Preparation of lysis buffer plates and single-cell samples]
FACS sort single cells into the plate. Immediately centrifuge ( for  at ) plates containing cells and keep them chilled on ice or at  until further processing.
Centrifuge: 1000 33
4 °C
-80 °C
When FACS sorting, take care of plate calibration/priming prior to single-cell deposition. If many plates are deposited at the same time, repeat the calibration/priming at least every 8 plates.We recommend defrost lysis buffer plates on ice prior to cell sorting, centrifuge (  for   at  ) and keep chilled on ice.Samples can be stored at   for at least 6months.
?. [Preparation of RNA Elution buffer]
Prepare the RNA elution buffer fresh for each experiment, as described below. We would recommend noting the lot numbers for reagents and plastics for every run, as this can be useful in troubleshooting. Keep the RNA elution buffer chilled until use.  ABC1ReagentPer well (μl)Final concentration2100 μM oligo dT&nbsp;0.152.5 μM310 mM dNTP1.52.5 mM4RNase Inhibitor (40U/μl)0.1510 U/μl5Nuclease-free water4.26Total6
ABC1ReagentPer well (μl)Final concentration2100 μM oligo dT&nbsp;0.152.5 μM310 mM dNTP1.52.5 mM4RNase Inhibitor (40U/μl)0.1510 U/μl5Nuclease-free water4.26Total6
?. Depending on the lysis buffer used, continue with option RLT buffer treated cells (A) or Triton buffer treated cells (B). We recommend introducing the non-template and multicell (10-50 cells) controls and locate them in the middle of the plate.A. RLT buffer treated cells: Single-cell RNA cleaning with RNAClean XP beadsi. Allow RNAClean XP beads to warm up to room temperature for  before use, gently mixing to ensure that the beads are evenly resuspended. ii. Dispense  of RNAClean XP beads into a clean 96-well Bead Plate with Eppendorf Multi-dispenser.iii. Seal the plate and centrifuge it ( for  at) to remove air bubbles in the wells.iv. Thaw and centrifuge ( for  at)  plate containing lysed cells from Step 2. Add additional RNAse-free water to the samples if required to a final volume of . v . Add  of RNAClean XP beads to each well of the working plate from step iv at room temperature, and mix thoroughly by pipetting up and down on the Zephyr instrument. Allow the mixture to stand for  at room temperature.vi. Move the plate to a low-elution magnet, and allow the beads to settle for  or until the solution is clear.vii. Once the RNAClean XP beads have settled, carefully remove and discard the supernatant without disturbing the beads. Keeping the 96-well plate on the magnet, wash the RNAClean XP beads with  of freshly prepared  (vol/vol) ethanol for , then remove and discard the ethanol wash. Do not disturb the beads.viii. Repeat Step vii once more.ix.Dispense  of RNA elution buffer from step 3 to each well of a new clean 96-well plate and place it on a chilled position of the robot deck. Add of RNA elution buffer to the RNAClean XP beads with purified single-cell RNA using the Zephyr.CRITICAL STEP Take the plate that contains beads with RNA and elution buffer under the hood and manually resuspend the beads in the RNA elution buffer with low-bind 10 μl tips with a multichannel pipette to facilitate the resuspension. Make sure that all beads are removed from the tips.x. Seal the plate and centrifuge it ( for  at ).xi. Proceed directly to the denaturation step 5.B.Triton Buffer treated cellsi. Thaw and centrifuge  ( for  at ) plate containing lysed single cells from Step2.ii. Dispense of freshly prepared RNA elution buffer from step 3 to each well of 96-well plate from step i. Seal the plate and centrifuge it  ( for  at ).Proceed directly to the denaturation step 5.
RLT buffer should be removed prior to subsequent reverse transcription.
25 µl
Centrifuge: 1000 33
4 °C
Keep bead plate covered with a plate seal on a shaker between cleaning procedures if there are many plates to be processed.
Centrifuge: 1000 33
4 °C
10 µl
We perform all purification steps using a Zephyr automated liquid-handling robot. Expose Zephyr deck to UV light for at least  . For the csRNA cleaning procedures use Zephyr 150 μl Barrier Sterile tips.
22 µl
100 µl
6 µl
5 µl
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
5 µl
Centrifuge: 1000 33
4 °C
?. [RNA denaturation]
Perform RNA denaturation on the thermal cycler with the heated lid set to 100 °C.  ABC1TemperatureTimeCycles272 ⁰C3 min134 ⁰Cforever
ABC1TemperatureTimeCycles272 ⁰C3 min134 ⁰Cforever
?. [RNA denaturation]
When the temperature reaches , immediately transfer plate with denatured single-cell RNA onto ice and proceed to the reverse transcription immediately.
4 °C
?. [Reverse Transcription]
Prepare the Reverse Transcription (RT) Master mix, as described below. We used this protocol for two reverse transcriptases, SmartScribe (SMT) and Maxima -H (Maxima), with enzymes corresponding to Master Mix compositions.
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							RT Master mix for Maxima enzyme   ABC1Reagent&nbsp;Per well (μl)Final concentration2Maxima buffer (5x)22.5x3Betaine (5 M)21.5 M4MgCl2 (100 mM)0.913 mM5TSO (10 μM)1.01.5 μM6RNase inhibitor (40 U/μl)0.2510 U/μl7Maxima RNaseH-minus RT (200 U/μl)0.120 U/μl8H2O0.759Total7.0RT Master mix for SMT enzyme  ABC1Reagent&nbsp;Per well (μl)Final concentration2Smartscribe buffer(5x)22.5x3DTT (100mM)0.58.5 mM4Betaine (5M)21.5 M5MgCl2 (1M)0.090.013 M6TSO (100 μM)0.11.8&nbsp; μM7RNase inhibitor (40U/ μl)0.251.76 U/μl8SmartScribe (100U/ μl)0.510 U/μl9H2O0.5610Total5.7
ABC1Reagent&nbsp;Per well (μl)Final concentration2Maxima buffer (5x)22.5x3Betaine (5 M)21.5 M4MgCl2 (100 mM)0.913 mM5TSO (10 μM)1.01.5 μM6RNase inhibitor (40 U/μl)0.2510 U/μl7Maxima RNaseH-minus RT (200 U/μl)0.120 U/μl8H2O0.759Total7.0
ABC1Reagent&nbsp;Per well (μl)Final concentration2Smartscribe buffer(5x)22.5x3DTT (100mM)0.58.5 mM4Betaine (5M)21.5 M5MgCl2 (1M)0.090.013 M6TSO (100 μM)0.11.8&nbsp; μM7RNase inhibitor (40U/ μl)0.251.76 U/μl8SmartScribe (100U/ μl)0.510 U/μl9H2O0.5610Total5.7
?. [Reverse Transcription]
Dispense  of Maxima RT master mix or  of SMT RT master mix to the plate with denatured RNA from step 6. Seal the plate, vortex on a medium speed and centrifuge it  ( for  at ).
7 µl
5.7 µl
Centrifuge: 1000 33
4 °C
?. [Reverse Transcription]
Reverse transcription will convert polyA (+) mRNA to cDNA. Place the plate in the thermal cycler and perform the following steps on a thermal cycler:Reverse Transcription conditions for Maxima RT  ABC1TemperatureTimeCycles250 ⁰C90 min1385 ⁰C5 min144 ⁰CHold1Reverse Transcription conditions for SMT RT  ABC1TemperatureTimeCycles242 ⁰C90 min1350 ⁰C2 min10442 ⁰C2 min570 ⁰C15 min164 ⁰Chold1
ABC1TemperatureTimeCycles250 ⁰C90 min1385 ⁰C5 min144 ⁰CHold1
ABC1TemperatureTimeCycles242 ⁰C90 min1350 ⁰C2 min10442 ⁰C2 min570 ⁰C15 min164 ⁰Chold1
?. [Reverse Transcription]
Collect the plate from the thermal cycler, centrifuge it  ( for  at ) and keep chilled on ice.
Centrifuge: 1000 33
4 °C
?. [PCR Amplification]
Prepare the PCR amplification Master mix, as described below.PCR amplification master mix for Maxima enzyme  ABC1Reagent&nbsp;Per well (μl)Final concentration2ISPCR primer (10 μM)0.50.4 μM3KAPA HiFi Hot Start Ready Mix (2x)12.52x4H2O1.05Total14PCR amplification master mix for SMT enzyme  ABC1Reagent&nbsp;Per well (μl)Final concentration2ISPCR primer (10 μM)0.250.3 μM3KAPA HiFi Hot Start Ready Mix (2x)12.52x4H2O2.255Total15
Identify the number of PCR cycles for each investigated cell type to avoid under- or over-amplification.
ABC1Reagent&nbsp;Per well (μl)Final concentration2ISPCR primer (10 μM)0.50.4 μM3KAPA HiFi Hot Start Ready Mix (2x)12.52x4H2O1.05Total14
ABC1Reagent&nbsp;Per well (μl)Final concentration2ISPCR primer (10 μM)0.250.3 μM3KAPA HiFi Hot Start Ready Mix (2x)12.52x4H2O2.255Total15
?. [PCR Amplification]
Dispense  of PCR master mix for Maxima RT or  of PCR master mix for SMT RT to each sample on the 96-well plate from step 10 using a Mantis. Seal the plate, vortex on a medium speed and centrifuge it  ( for  at ). Place the plate in the thermal cycler and perform the following steps:PCR amplification protocol for Max RT   ABC1TemperatureTimeCycles298 ⁰C3 min1398 ⁰C15 sec20 / 22 / 25 cycles depending on the cell type467 ⁰C20 sec572 ⁰C6 min672 ⁰C6 min174 ⁰CholdPCR amplification protocol for SMT RT  ABC1TemperatureTimeCycles298 ⁰C3 min1398 ⁰C20 sec20 / 22 / 25 cycles depending on the cell type467 ⁰C15 sec572 ⁰C6 min672 ⁰C5 min174 ⁰Chold 
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14 µl
15 µl
Centrifuge: 1000 33
4 °C
ABC1TemperatureTimeCycles298 ⁰C3 min1398 ⁰C15 sec20 / 22 / 25 cycles depending on the cell type467 ⁰C20 sec572 ⁰C6 min672 ⁰C6 min174 ⁰Chold
ABC1TemperatureTimeCycles298 ⁰C3 min1398 ⁰C20 sec20 / 22 / 25 cycles depending on the cell type467 ⁰C15 sec572 ⁰C6 min672 ⁰C5 min174 ⁰Chold
Amplified cDNA samples can be stored in   for several months before purification.
?. [PCR Amplification]
Proceed with the Agencourt AMPure beads for cleaning on the Zephyr instrument, or keep the plate at  if continuing the next day, or  for long term storage.
4 °C
-20 °C
?. [Purification of the amplified cDNA]
Allow Agencourt AMPure XP beads to warm up to room temperature for  before use, gently mixing to ensure that the beads are evenly resuspended. Dispense  of Agencourt AMPure XP beads into the clean 96-well Bead Plate with Eppendorf Multi-dispenser.
Steps 14–25should be performed in a dedicated post-amplification room.
25 µl
?. [Purification of the amplified cDNA]
Centrifuge  ( for  at ) the plate containing the amplified cDNA from Step13.
Centrifuge: 1000 33
4 °C
?. [Purification of the amplified cDNA]
Add  of the Agencourt AMPure XP beads to each well of the 96-well plate containing the PCR-amplified cDNA molecules at room temperature, and mix thoroughly by pipetting up and down. Allow the mixture to stand for  at room temperature.
25 µl
?. [Purification of the amplified cDNA]
Move the plate to a magnet, and allow the beads to settle for or until the solution is clear.
?. [Purification of the amplified cDNA]
Carefully remove and discard the supernatant without disturbing the beads, once the beads have settled.
?. [Purification of the amplified cDNA]
Keeping the 96-well plate on the magnet, wash the beads with  of freshly prepared  (vol/vol) ethanol for , and then remove and discard the ethanol wash. Do not disturb the beads.
100 µl
?. [Purification of the amplified cDNA]
Repeat Step 19 once more.
?. [Purification of the amplified cDNA]
Remove and discard any remaining ethanol solution from the well without disturbing the beads, and then allow the beads to dry for ~.
?. [Purification of the amplified cDNA]
Add  of nuclease-free water to the beads, remove the plate from the magnet and resuspend by pipetting up and down at room temperature.
25 µl
?. [Purification of the amplified cDNA]
Incubate this 96-well plate for  off the magnet at room temperature.
?. [Purification of the amplified cDNA]
Return the 96-well plate to the magnet, and allow the beads to settle for  or until the solution is clear.
?. [Purification of the amplified cDNA]
Transfer the supernatant, which contains the purified cDNA to a new 96-well plate at room temperature without disturbing the beads.
The amplified cDNA can be stored at   for >6 months before library preparation.
?. [Quality Control of Amplified cDNA and input normalization]
We use an Agilent Bioanalyzer for a cDNA QC on a high-sensitivity chip. Run  of the eluted sample on a high-sensitivity chip on the Agilent. Dilute the library to 1 in 10 if cDNA is highly concentrated. It is important to quantify cDNA concentration accurately. Overestimation can affect the normalization of the cDNA input for the Nextera library preparation and lead to skewed Nextera libraries profiles.
1 µl
?. [Sample normalization]
For cDNA input normalization, we check 11 randomized wells throughout a plate (A6; B4; B11; C3; D4; D12; F2; F9; G3; G8; H5). Measure cDNA concentration between 200-8000bp in all 11 samples, and find an average concentration that will be applied to the whole plate. Remove the outliers for accurate template normalization. The final average concentration of cDNA should be normalized to 0.3 ng/μl for optimal tagmentation. Normalize multicell controls separately.
Steps 27–29 should be performed in a dedicated post-amplification room. Illumina libraries are prepared using the tagmentation approach with Nextera XT DNA Library Prep Kit (Illumina). We recommend following the manufacturer’s instructions for the applied kit.
?. [Sample normalization]
Sample normalization is performed on the Zephyr instrument. A fixed amount of cDNA will be used for the dilution. Take  of cDNA from the individual wells and dilute with water to achieve a final concentration of 0.3 ng/ul.
5 µl
?. [Sample normalization]
Seal the normalised plate and centrifuge it  ( for  at ), vortex on medium speed for  and centrifuge  ( for  at ) again.
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
Normalised cDNAcan be stored at   for continuation the next day, or at   for a week.
?. [Library construction using the Nextera XT Library Preparation Kit]
Transfer  of normalised samples from step 29 using the Zephyr instrument to the working Nextera Plate. Keep plates chilled and continue with the Nextera protocol.
1.2 µl
?. [Library construction using the Nextera XT Library Preparation Kit]
Prepare the Nextera master mix, as described below and dispense with the Mantis using a Low Volume (LV) chip.
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							  AB1Reagent&nbsp;Per well (μl)2Tagmentation DNA buffer2.53Amplification Tagment Mix1.254Total3.75
AB1Reagent&nbsp;Per well (μl)2Tagmentation DNA buffer2.53Amplification Tagment Mix1.254Total3.75
?. [Library construction using the Nextera XT Library Preparation Kit]
Seal the plate and centrifuge  ( for  at ), vortex on medium speedfor  and centrifuge  ( for  at ) again.
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
?. [Library construction using the Nextera XT Library Preparation Kit]
Place the Nextera plate in a thermal cycler and run the following program:
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							  AB1TemperatureTime255 ⁰C10min34 ⁰CHold
AB1TemperatureTime255 ⁰C10min34 ⁰CHold
?. [Library construction using the Nextera XT Library Preparation Kit]
Once the temperature reaches , remove from the thermal cycler and centrifuge  ( for  at ) the plate.
4 °C
Centrifuge: 1000 33
4 °C
?. [Library construction using the Nextera XT Library Preparation Kit]
Wash Mantis LV chip with water by dispensing 50-100 μl of water into the waste plate.
?. [Library construction using the Nextera XT Library Preparation Kit]
Dispense  NT buffer into each well to neutralize the reaction.
1.2 µl
?. [Nextera Index preparation]
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							Seal the plate and centrifuge  ( for  at ), vortex on medium speedfor 1min and centrifuge  ( for  at ) again.
Prepare the Indexes set mix (A-B-C-D) in 96-well Index Plate according to the manufacturer’s instructions. Defrost and spin down Index 1(i7) and Index 2 (i5). Arrange the index primers in the Index Plate Fixture as follows: Index 1 (i7) adapters in columns 1-12 in ascending or descending order. Index 2 (i7) adapters in columns A-H in ascending or descending order(Figure S3).  Using a multichannel pipette, add  of i7 adapters down each row. Replace the vial cap with a new cap. Using a multichannel pipette, add   of i5 adapters down each column. Replace the vial cap with the new cap.
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
?. [Nextera Index preparation]
Keep premade plates with index barcodes chilled until use or in the freezer at  for long term storage.
-20 °C
?. [Nextera Index preparation]
Dispense  of Index Adapters on the Zephyr instrument to the working Nextera plate from step 36. Seal the plate and centrifuge  ( for  at ).
2.5 µl
Centrifuge: 1000 33
4 °C
?. [Nextera Library amplification]
40.Wash Mantis LV chip by dispensing 50-100 μl of water into the waste plate.
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?. [Nextera Library amplification]
Dispense  of NPM buffer by the Mantis dispenser to each well of the working Nextera plate.
3.7 µl
?. [Nextera Library amplification]
Seal the plate and centrifuge  ( for  at ), vortex on medium speed for 1min and centrifuge  ( for  at ) again.
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
?. [Nextera Library amplification]
Place the library prep plate in a thermal cycler and perform the following program:   ABC1TemperatureTimeCycles272 ⁰C3 min1395 ⁰C30 sec1495 ⁰C10 sec&nbsp;12555 ⁰C30 sec672 ⁰C30 sec772 ⁰C&nbsp;&nbsp;5 min184 ⁰CHold1
ABC1TemperatureTimeCycles272 ⁰C3 min1395 ⁰C30 sec1495 ⁰C10 sec&nbsp;12555 ⁰C30 sec672 ⁰C30 sec772 ⁰C&nbsp;&nbsp;5 min184 ⁰CHold1
?. [Nextera Library amplification]
Once the temperature reaches , remove from the thermal cycler. Centrifuge the plate  ( for  at ), vortex on medium speed for 1min and centrifuge  ( for  at ) again.
4 °C
Centrifuge: 1000 33
4 °C
Centrifuge: 1000 33
4 °C
?. [Nextera library pooling and purification]
Collect of each sample (except the Control wells) into 8-well PCR strip tubes using a multichannel pipette. Pool control wells if these samples are used for the analysis.
5 µl
?. [Nextera library pooling and purification]
Then pool amplified libraries from PCR strips into a single 1.5 ml vial (≈). Briefly spin down the strips and collect the rest of the samples.
595 µl
?. [Nextera library pooling and purification]
Measure the sample volume with the pipette and add Agencourt AMPure XP beads at 1:1 ratio. Mix well by pipetting. Incubate for  at room temperature.
?. [Nextera library pooling and purification]
Place the vial with the beads/sample mix on the magnet for  or wait till the solution is clear.
?. [Nextera library pooling and purification]
Carefully remove and discard the supernatant without disturbing the beads, once the beads have settled.
?. [Nextera library pooling and purification]
Keeping the vial on the magnet, wash the beads with  of freshly prepared  (vol/vol) ethanol for , and then remove and discard the ethanol wash. Do not disturb the beads.
100 µl
?. [Nextera library pooling and purification]
Repeat Step 50 once more.
?. [Nextera library pooling and purification]
Remove and discard any remaining ethanol solution from the well without disturbing the beads, and then allow the beads to dry for ~. Leave the vial cap open to air-dry the beads.
?. [Nextera library pooling and purification]
Remove the vial from the magnet, add  of nuclease-free water to beads and resuspend by pipetting up and down at room temperature.
100 µl
?. [Nextera library pooling and purification]
Incubate the vial for  off the magnet at room temperature.
?. [Nextera library pooling and purification]
Return the vial to the magnet, and allow the beads to settle for or until the solution is clear.
?. [Nextera library pooling and purification]
Transfer the supernatant, which contains the purified Nextera libraries to a new vial without disturbing the beads.
?. [Nextera library pooling and purification]
Keep the samples at  if continued the next day or  for long term storage.
4 °C
-20 °C
?. [Quality Control of Nextera sequencing libraries and normalization for sequencing]
Run of the eluted sample from step 57 in triplicate on an Agilent Bioanalyser using a DNA 1000 assay chip or 1 in 10 diluted sample on a high-sensitivity chip.
1 µl
?. [Quality Control of Nextera sequencing libraries and normalization for sequencing]
Note the molarity of each reading and take an average of the 3 triplicates discarding any outliers.
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?. [Quality Control of Nextera sequencing libraries and normalization for sequencing]
Taking the molarity reading from step 59. In a fresh 1.5 ml tube combine the calculated volume of the amplified library pool with nuclease-free water to produce a final sequencing library pool of 2.8 nM.
?. [Quality Control of Nextera sequencing libraries and normalization for sequencing]
In a fresh 1.5 ml tube equimolar pool the 2.8 nM sequencing library from step 60 with 3 additional sequencing library pools which have been processed through this workflow. To produce a final sequencing pool containing 384 samples.
?. [Library sequencing]
Libraries were submitted for sequencing on an Illumina HiSeq 4000 chemistry (paired-end, 75-bp reads). Perform single-end (SE) or paired-end sequencing (PE) of the Single-cell derived libraries according to the manufacturer’s protocol. Alternative sequencing options to achieve comparable sequencing depth are; the NextSeq 550 with high output flow cell or NovaSeq 6000 SP flow cell on one lane of an XP workflow.