Feb 19, 2020

Public workspaceSmart-seq3 Protocol V.3

DOI
dx.doi.org/10.17504/protocols.io.bcq4ivyw
  • 1Karolinska Institute Stockholm
  • Human Cell Atlas Method Development Community
  • single cell transcriptome
Michael Hagemann-Jensen
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DOI: dx.doi.org/10.17504/protocols.io.bcq4ivyw
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.bcq4ivyw
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: February 19, 2020
Last Modified: February 19, 2020
Protocol Integer ID: 33276
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:
OligoVendorPurificationWorking concentrationSequence
Smartseq3_OligodT30VNIDTHPLC100uM/5Biosg/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN
Smartseq3_N8_TSOIDTRNase-Free HPLC100uM/5Biosg/AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG
Fwd_PCR_primerIDTHPLC100uMTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAA*T*G
Rev_PCR_primerIDTHPLC100uMACGAGCATCAGCAGCATAC*G*A
* 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

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.


Materials
MATERIALS
ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML
ReagentAgilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
ReagentNextera XT DNA Library Preparation KitilluminaCatalog #FC-131-1096
ReagentQIAquick Gel Extraction KitQiagenCatalog #28704
ReagentDNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
ReagentRecombinant RNAse InhibitorTakarabioCatalog #2313A
ReagentDithiothreitol (DTT)Thermo Fisher ScientificCatalog #707265ML
ReagentdNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977035
ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
ReagentSDS, 10% Solution, RNase-freeThermo FisherCatalog #AM9822
ReagentMaxima H Minus Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0751
ReagentPoly Ethylene Glycol (PEG) 8000Sigma AldrichCatalog #89510-250G-F
ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
ReagentMagnesium Chloride (1M Solution)Invitrogen - Thermo FisherCatalog #AM9530G
ReagentGTP (Tris buffered solution 100mM)Thermo ScientificCatalog #R1461
ReagentTrizma-baseSigma AldrichCatalog #T6791-100G
ReagentKAPA HiFi Hotstart PCR kitRocheCatalog #KK2502
ReagentPhusion High-Fidelity DNA Polymerase (2 U/µL) Thermo ScientificCatalog # F530L
ReagentSera-Mag Speed BeadsGe HealthcareCatalog #65152105050250
ReagentSodium AzideSigma AldrichCatalog #S2002-100G
ReagentIGEPAL® CA-630 Sigma AldrichCatalog #I8896
ReagentArmadillo PCR Plate 96-well clear semi-skirted white wellsThermo ScientificCatalog #AB3596
ReagentArmadillo PCR Plate 384-wellThermo ScientificCatalog # AB2384B
ReagentQuantiFluor® dsDNA SystemPromegaCatalog #E2670
ReagentE-Gel™ EX Agarose Gels 2% Thermo ScientificCatalog #G402002
Reagent2-propanolSigma AldrichCatalog #I9516
ReagentNN-DimethylformamideSigma AldrichCatalog #D4551
Before starting
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 TemperatureOn 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
Prepare lysis plates
Prepare lysis buffer mix

ReagentReaction conc.uL per. reaction96 well plate384 well plate
Guanidine Hydrochloride (8000mM ; Optional)0mM - 50mM0.00--
Poly-ethylene Glycol 8000 (50% solution)5%0.4044164
Triton X-100 (10% solution)0.1%0.033.312.3
ERCC spike-ins (Optional)----
RNAse Inhibitor (40u/uL)0.5u/uL0.044.115.4
OligodT30VN (100uM)0.5uM0.022.28.2
dNTPs (25mM/each)0.5mM/each0.088.832.8
Nuclease Free Water2.43267.6 997.3
Total3 uL330 uL1230 uL
Reaction concentrations for PEG8000, OligodT30VN and dNTPs, are adjusted to and reflect their concentration in the reverse transcription reaction (4uL)

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 Amount3 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
Sample collection
Sort single cells into Amount3 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 Temperature-80 °C freezer or dry-ice.

Cell lysis
Cell lysis
Remove the plate of sorted cells from the -80 freezer and incubate in a thermocycler with heated lid at Temperature72 °C for Duration00:10:00 , followed by a Temperature4 °C hold (keeping the storage seal sheet on the plate, unless damaged or loose).

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

ReagentReaction conc.uL per. reaction96 well plate384 well plate
Tris-HCl pH 8.3 (1M)25mM0.11141
NaCl (1M)30mM0.1213.249.2
MgCl2 (100mM)2.5mM0.11141
GTP (100mM)1mM0.044.416.4
DTT (100mM)8mM0.3235.2131.2
RNase Inhibitor (40u/uL)0.5u/uL0.055.520.5
TSO (100uM)2uM0.088.832.8
Maxima H-minus RT enzyme (200U/uL)2u/uL0.044.416.4
Nuclease Free Water0.1516.561.5
Total1uL110uL410uL

Add Amount1 µL 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;

42 °C90 min1x
50 °C2 min10x
42 °C2 min
85 °C5 min1x

Preamplification PCR
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.

ReagentReaction conc.uL per. reaction96 well plate384 well plate
Kapa HiFi HotStart buffer (5X)1X2.0220820
dNTPs (25mM/each)0.3mM/each0.1213.249.2
MgCl2 (100mM)0.5mM0.055.520.5
Fwd Primer (100uM)0.5uM0.055.520.5
Rev Primer (100uM)0.1uM0.011.14.1
Polymerase (1U/uL)0.02U/uL0.22282
Nuclease Free Water3.57392.71463.7
Total6uL660uL2460uL

AddAmount6 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.


StepTemperatureTimeCycles
Initial denaturation98 °C3 min1x
Denaturation98 °C20 sec18-25x
Annealing65°C30 sec
Elongation72 °C4 min
Final Elongation72 °C5 min1x
Hold4 °CHold
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)
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
CITATION
Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard. mcSCRB-seq protocol. Nature Communications.
LINK
dx.doi.org/10.17504/protocols.io.p9kdr4w

ReagentAmount
PEG 800011 g
NaCl, 5M 10 mL
Tris-HCL, 1M, pH 8.0 500 μL
EDTA, 0.5M 100 μL
IGEPAL, 10% solution 50 μL
Sodium Azide, 10% solution 250 μL
UltraPure Waterup to 49 mL
Total 49 mL

Add all ingredients into a Amount50 mL falcon tube, but do not add the total amount of water until after PEG is completely solubilized.

Incubate at Temperature40 °C and vortex regularly until PEG is completely dissolved.

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

Pipette Amount1000 µL of bead suspension into a 1.5 mL tube.

Place on magnet stand Remove supernatant.

Add Amount1000 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 Amount900 µL 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads.

Add to PEG solution above and mix well.
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 TemperatureRoom temperature for Duration00:08:00 .

3. Place on magnet and allow beads to settle. Roughly Duration00:05:00 .

4. Discard supernatant, and wash once with Amount20 µL / Amount100 µL of freshly prepared 80% Ethanol for 384 / 96 well plates respectively.

5. Remove Ethanol and let the beads air dry for Duration00:02:00 - Duration00:05:00

6. Elute cDNA in Amount12 µL UltraPure Water, resuspend beads and incubate for Duration00:05:00 .

DNA concentration measurement and normalization (Optional, but recommended)
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 Amount49 µL /Amount99 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 Amount1 µL of cDNA to each well. Incubate asssay for Duration00:05:00 at TemperatureRoom temperature

7. Use a plate reader, to measure fluorescence (504nM Excitation/ 531nM Emission) 8. Calculate cDNA concentration.

9. Calculate water needed to dilute Amount1 µL cDNA toConcentration100 pg/uL .
1. Prepare normalization plate by adding the calculated water volumes to each well.

2. Add Amount1 µL of preamplified cDNA to each well.

Quality Control check (Optional)
Quality Control check (Optional)
Check the cDNA preamplification library content and quality on a Agilent Bioanalzyer, using High Sensitivity DNA Analysis chips.
Expected result
Example of one HEK cell.


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

ReagentAmount (uL)Concentration in 4X
Tris-HCl pH 7.5 (1M)4040mM
MgCl2 (100mM)20020mM
Dimethylformamide (DMF)20020%
UltraPure Water560
Total1000 uL

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..

ReagentReaction conc.uL per. reaction96 well plate384 well plate
Tagmentation buffer (4x)1X0.555205
Amplicon Tagmentation Mix (Tn5)0.088.832.8
UltraPure water0.4246.2172.2
Total1uL110uL410uL

3. Dispense Amount1 µL of Tagmentation mix to a new 96 or 384 well plate.

4. Add Amount1 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 Temperature55 °C for Duration00:10:00 .

6. To strip off the Tn5 from the DNA, add Amount0.5 µL of 0.2% SDS to each well. Centrifuge quickly and incubate for Duration00:05:00 .

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 Amount1.5 µL Nextera Index primers to each well as follows
ReagentReaction conc.uL per. reaction
Custom S50X index primer (0.5uM)0.1uM0.75
Custom N70X index primer (0.5uM)0.1uM0.75


9. Prepare Tagmentation PCR mix.

ReagentReaction conc.uL per. reaction96 well plate384 well plate
Phusion HF buffer (5X)1X1.4154574
dNTPs (25mM/each)0.2mM/each0.066.223
Phusion HF (2U/uL)0.01U/uL0.043.914.4
H2O1.51166618.7
Total3uL330uL1230uL

10. Add Amount3 µL of Tagmentation PCR mix to each well, centrifuge quickly and incubate in a thermocycler using the following PCR program.

StepTemperatureTimeCycles
Gap-filling72 °C3 min1x
Initial denaturation98 °C3 min1x
Denaturation98 °C10 sec12x
Annealing55 °C30 sec
Elongation72 °C30 sec
Final Elongation72 °C5 min1x
Hold4 °CHold


Library clean-up
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 Duration00:08:00 at TemperatureRoom temperature

2. Place on magnet and allow beads to settle. Roughly Duration00:05:00 .

4. Discard supernatant, and wash twice with >= Amount1000 µL freshly prepared 80% Ethanol.

5. Remove Ethanol and let the beads air dry for at least Duration00:05:00

6. Elute cDNA in Amount40 µL UltraPure Water, resuspend beads and incubate for Duration00:05:00 .

(Optional) Size selection via Gel-cutting and extraction
(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 Amount20 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 Duration00:12:00

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
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.

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


Use Qubit fluorometer or similar to quantify the library.

Calcutate the final library concentration, using above metrics.

Sequencing
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
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).
Software
bcl2fastq
NAME
Illumina
DEVELOPER
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: 2x150bp 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.
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.
LINK
https://doi.org/10.1093/gigascience/giy059

Software
zUMIs
NAME
Linux
OS
https://github.com/sdparekh/zUMIs
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:
Command
Smartseq3.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: ~
  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
  velocyto: 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


Now, simply start zUMIs with the following command:
Command
Invoke zUMIs
zUMIs-master.sh -y Smartseq3.yaml


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