TITLE: SMART-Seq
AUTHORS: cecilia  , Suzie Alarcon, Alessandro  Sette

[DESCRIPTION]
This protocol details the cell lysis / Oligot-dT priming, reverse transcription, PCR preamplification and quality Check cDNA and tagmentation reaction.

[STEPS]
SECTION: Cell Lysis / Oligot-dT Priming
1. 
 
Dilute the oligo-dT30VN primer to 10 Micromolar (µM) by adding 10 µL of 100 Micromolar (µM) oligo-dT primers and 90 µL of nuclease-free water to a tube and mix well.

SECTION: Cell Lysis / Oligot-dT Priming
2. Prepare cell lysis buffer by adding 1 µL of RNase inhibitor to 19 µL of a 0.2% (vol/vol) Triton X-100 solution.
 

SECTION: Cell Lysis / Oligot-dT Priming
3. Isolate single cells in the lowest possible volume (preferably ≤0.5 µL, possibly 0.3 µL) or pipet the appropriate amount of RNA into a 0.2 mL thin-walled PCR tube. Single cells can be obtained either by using a micro capillary pipette or via FACS.

SECTION: Cell Lysis / Oligot-dT Priming
4. Place each single cell into a 0.2 mL thin-walled PCR tube containing 2 µL of cell lysis buffer, 1 µL of oligo-dT primer and 1 µL of dNTP mix.

SECTION: Cell Lysis / Oligot-dT Priming
5. Quickly vortex the tube to mix, and then spin down the solution (700 g for 10 s at Room temperature) and immediately place it on ice.

SECTION: Cell Lysis / Oligot-dT Priming
6. Incubate the samples at 72 °C for 3 min and immediately put the tube back on ice.

SECTION: Cell Lysis / Oligot-dT Priming
7. Spin down the samples (700 g for 10 s at 72 Room temperature) to collect the liquid at the bottom of the tubes, and then put them immediately back on ice.
 
Purified RNA:

Xul        RNA up to 2.5ul
1ul        oligo-dT Primer (10uM)
1ul        dNTP (10mM)
xul        H2O
4.5ul    Total

72 °C for 3 min, snap cool.

SECTION: Reverse Transcription
8. Prepare the RT mix for all reactions plus one additional reaction by combining and mixing the reagents listed in the table below.
 
     Component      Volume (ul)      Final Conc        Superscipt II      0.50      100 U        RNAse inhibitor (40 U/ul)      0.25      10   U        Superscript II FS buffer (5X)      2.00      1X        DTT (100 mM)      0.50      5   mM        Betaine (5 M)      2.00      1 M        MgCl2 (1 M)      0.06      6   mM        TSO (100uM)      0.10      1 uM        H2O      0.29      -        Total      5.70      -   
 
SECTION: Reverse Transcription
9. Add 5.7 µL of RT mix to Samples for a total of 10 µL.

SECTION: Reverse Transcription
10. Spin and incubate as follows:
 
     Cycle      Temperature   (°C)      Time      Purpose        1      42      90 min      RT and template-switching        2–11      50      2 min      Unfolding of RNA secondary structures               42      2 min      Completion/continuation of RT   and template-switching        12      70      15 min      Enzyme inactivation        13      4      Hold      Safe storage   
 
SECTION: PCR preamplification
11. Prepare the PCR mix for all reactions plus one additional reaction by combining and mixing the following components:
 
     Component      Volume   (μl)      Final   concentration        First-strand   reaction      10      –        KAPA HiFi HotStart ReadyMix (2×)      12.50      1×        IS   PCR primers (10 μM)      0.25      0.1 μM        Nuclease-free water      2.25      –        Total   volume      25      –   
 
SECTION: PCR preamplification
12. Add 15 µL of PCR mix to each tube from Step 12, which contains the first-strand reaction and perform the PCR in a thermal cycler by using the following program:
 
     Cycle      Denature      Anneal      Extend      Hold        1      98   °C, 3 min      –      –      –        2–19   (see below)      98 °C,   20 s      67 °C,   15 s      72 °C,   6 min      –        20      –      –      72   °C, 5 min      –        21      –      –      –      4 °C   
 
 
     Input Amount  Total RNA      Input Amount,  Cells      Typical No. of  PCR Cycles           10 ng      1,000 cells      12        1 ng      100 cells      12        500 pg      50 cells      13        100 pg      10 cells      15        10 pg      1 cell      18   

 
 

SECTION: Ampure Cleanup
13. Perform a typical Ampure cleanup using 1:1 ratio of Ampure:cDNA.
SECTION: Ampure Cleanup
14. Elute using 17.5 µL EB solution and pipette 15 µL to transfer to a new tube.

SECTION: Quality Check cDNA
15. Run a High Sensitivity Bioanalyzer Chip to check for quality of cDNA.
SECTION: Quality Check cDNA
16. A good library should be free of short (<500 bp) fragments and should show a peak at 1.5–2 kb.
  

SECTION: Tagmentation Reaction
17. Setup the tagmentation RXN as follows:

 
 
     Component      Volume   (μl)      Final   concentration        Tagment   DNA buffer (TD, 2×)      10      1×        Amplicon   tagment mix      5      –        DNA   from PCR      Variable      –        Nuclease-free   water      Variable      –        Total   volume      20      –   

 
 
SECTION: Tagmentation Reaction
18. Incubate in a thermal cycler at 55 °C for 5 min and bring to 4 °C HOLD.

SECTION: Tagmentation Reaction
19. Add 5 µL of NT buffer to the 20 µL RXN and mix.

SECTION: Tagmentation Reaction
20. Incubate at 4 Room temperature for 5 min.

SECTION: Enrichment of Tagmented cDNA
21. Prepare the following PCR RXN as follows:
 
     Component      Volume   (μl)        DNA      25        Nextera   PCR master mix      15        Index   1 primers (N7xx)      5        Index   2 primers (N5xx)      5        Total   volume      50   
 
SECTION: Enrichment of Tagmented cDNA
22. Run the PCR RXN on a thermal cycler with the following conditions:

 
 
     Cycle      Denature      Anneal      Extend      Hold        1      –      –      72   °C, 3 min      –        2      95 °C,   30 s      –      –      –        3–14*      95   °C, 10 s      55   °C, 30 s      72   °C, 30 s      –        15      –      –      72 °C,   5 min      –        16      –      –      –      4   °C   

 
 
SECTION: Expected results:
23.