TITLE: Part 1: SmartSeq
AUTHORS: cecilia  , Suzie Alarcon, Alessandro  Sette

[DESCRIPTION]
This protocol details the procedure of SmartSeq.

[STEPS]
SECTION: INPUT:
1. Use total RNA, ranging between 10 pg-30 pg up to 10 ng. 2.6 µL will be used per sample.

SECTION: ANNEAL PRIMERS:
2. on ice, add each sample (2.6 µL total RNA) to a thin-walled 0.2 mL PCR tube and add:
 
     Item      Volume   (uL)      ______xMM      Lot#        oligo-dT primer (10uM)      1                      dNTP mix (10mM)      1                 
 

SECTION: ANNEAL PRIMERS:
3. Centrifuge at 700 x g for 10 s at Room temperature. Incubate @72 °C for 3 min, spin down, put on ice.

SECTION: ANNEAL PRIMERS:
4. REVERSE TRANSCRIPTION: add the following on ice:
 
     Item      Volume(uL)      ______xMM      Lot#        SuperScriptII reverse transcriptase (200U/uL)      0.50                      RNAse inhibitor (40U/uL)      0.25                      SuperscriptII first-strand buffer (5x)      2                      DTT (100mM)      0.50                      Betaine(5M)*      2                      Nuclease-free H2O      0.06                      TSO(100uM)**      0.10                      Total volume:      5.41             N/A   
 

SECTION: ANNEAL PRIMERS:
5. Mix, spin down, and incubate in thermocycler on following settings:
 
     Step      Temp(C)      Time(hh:mm:ss)        1      42      01:30:00        2      50      00:02:00        3      42      00:02:00        4      Return to step 2, 5x               5      70      00:15:00        6      4      hold   
 
SECTION: ANNEAL PRIMERS:
6. PCR PRE-AMPLIFICATION: Prepare Master Mix during Reverse Transcription rxn. 
     Item      Volume(uL)      ______xMM      Lot#        First-strand rxn (previous step)      10             N/A        Kapa HiFi HotStart ReadyMix (2x)      12.5                      IS PCR primers (10uM)      0.25                      H2O      2.25                      Total volume:      25             N/A   
 
SECTION: ANNEAL PRIMERS:
7. Add 15 µL PreAmp Master Mix to each sample, seal, mix, spin down, and incubate in thermocycler on following settings:
 
     Step      Temp(C)      Time(hh:mm:ss)        1      98      00:03:00        2      98      00:00:20        3      67      00:00:15        4      72      00:06:00        5      Return to step 2      17x (18 cycles total)        6      72      00:05:00        7      4      hold   
 
SECTION: ANNEAL PRIMERS:
8. Do a standard Ampure or equivalent 1X (1:1 ratio bead to sample) Bead cleanup (add 25 µL beads to each sample).

SECTION: ANNEAL PRIMERS:
8.1. Allow beads to incubate with sample for 5 min at Room temperature. After 5 minutes, place samples on magnet until the supernatant is clear and all beads are on the wall of the tube. Carefully remove the supernatant and immediately move to wash steps.

SECTION: ANNEAL PRIMERS:
8.4. Elute with 17.5 µL H2O, keep 15 µL. Once beads are dry, resuspend in eluate and incubate for 2 min - 5 min. Place slurry on magnet and allow supernatant to clear. Once all beads are on the magnet, carefully remove 15 µL of the clear eluate.

SECTION: ANNEAL PRIMERS:
8.3. 80% EtOH wash 2. Once second EtOH wash is completed and EtOH has been removed from the beads, allow the beads to dry until they look glossy.
 

SECTION: ANNEAL PRIMERS:
8.2. Carefully pipette 150 µL-200 µL of freshly prepared 80% EtOH to the supernatant and incubate for 30 s. Remove EtOH and immediately repeat this step.

SECTION: ANNEAL PRIMERS:
9. Quant the cleaned cDNA on Tapestation, using D5000 reagents or HSD5000, depending on expected yield and according to manufacturers suggestions.