TITLE: Part 2: Custom QXT
AUTHORS: cecilia  , Suzie Alarcon, Alessandro  Sette

[DESCRIPTION]
This protocol details the procedure of Custom QXT.

[STEPS]
SECTION: A: Fragmentation and Adaptor-tagging
1. FIRST, IF POSSIBLE: Normalize cDNA samples to 2x the input mass (for 1 ng cDNA input will be 2 ng/10.13 µL, for the 5 ng input will be 10 ng/10.13 µL). Run quants (picogreen) to assess concentration. If normalization was successful, add one volume of water to the samples to bring it back to 1x concentration and continue with 10.16 µL.
 
      1ng cDNA input:  5ng cDNA   input:    ❏       INPUT: 1ng cDNA in   10.16uL water  ❏       INPUT: 5ng cDNA in   10.16uL water    ❏       QXT DILUTION: 1:20 dilution of QXT enzyme:Storage   Soln., at least 2uL of dilution/sample  ❏       QXT DILUTION: 1:6 dilution of QXT enzyme:Storage   Soln., at least 2uL of dilution/sample    ❏       MASTER MIX:  ❏       MASTER MIX:    Reagent  1 rxn (uL)  ____rxn  Reagent  1 rxn (uL)  ____rxn    cDNA  10.16  N/A  cDNA  10.16  N/A    5x TD Buffer  8.84     5x TD Buffer  8.84       QXTEnzyme  0.1     QXTEnzyme  0.33       StorageSoln  1.9     StorageSoln  1.67       Total:  21     Total:  21     
 
 
 

SECTION: A: Fragmentation and Adaptor-tagging
2. Place samples in thermocycler on “DNA Fragmentation” program:
 
     Step      Temp(C)      Time(hh:mm:ss)        1      55      00:10:00        2      4      00:01:00        3      4      Hold   
 
SECTION: A: Fragmentation and Adaptor-tagging
3. Add 5 µL 0.2%SDS and 24 µL water/sample.
 
     Reagent      1   rxn (uL)      ____rxn   (uL)        0.2%SDS      5               H2O      24               Totals:      29          
 

SECTION: A: Fragmentation and Adaptor-tagging
4. Seal, vortex, incubate at Room temperature for 1 min.

SECTION: B: Ampure bead purification 1
5.  
        Single cleanup  Double   SPRI cleanup (experimental)    ❏       Vortex beads and incubate @ RT for 30minutes   ❏     Vortex beads and equilibrate @ RT   for 30minutes     ❏       Add 49uL bead mix/sample (1.0x)  ❏     0.5x cleanup (>600bp fragment   removal): Add 25uL beads to each 50uL sample, vortex, briefly spin     ❏       Seal samples, vortex, briefly spin down  ❏     Incubate samples 5 mins @RT    ❏       Incubate samples @RT 5 minutes  ❏     Add samples to magnet rack, allow   solution to clear, save supernatant to new clean tube (optional: save beads)    ❏       Put samples on magnet rack, allow to clear, discard supernatant  ❏     0.2x cleanup (removal of   <150bp fragments, rounding out total cleanup to 0.7x): Add 10uL bead   mix/sample, seal, mix, incubate for 5 mins @RT    ❏       Wash 1 with 200uL 80% EtOH  ❏     Put samples on magnet rack, allow   to clear, discard supernatant    ❏       Wash 2 with 200uL 80% EtOH  ❏     Wash 1 with 200uL 80% EtOH    ❏       Remove last of the EtOH, allow samples to air dry for at least 10 minutes.  ❏     Wash 2 with 200uL 80% EtOH    ❏       Elute with 24uL H2O, incubate for 2 mins @RT  ❏     Remove the last Ethanol and allow   samples to air dry for at least 10 minutes      ❏     Elute with 24uL H2O, incubate for   2 mins @RT  
 
SECTION: C: Amplification
6. Add 24 µL Kapa Hotstart Master Mix/sample.

SECTION: C: Amplification
7. Add 1 µL i7 primer and 1 µL i5 indexing primer, or 2 µL from a multiplex plate.
 


SECTION: C: Amplification
8. Seal, vortex, briefly spin down samples, put in thermocycler on “Pre-Capture PCR” protocol. 
        Step  Temp(C)  Time(hh:mm:ss)      1  68  00:02:00      2  98  00:02:00      3  98  00:00:30      4  57  00:00:30      5  72  00:01:00      6  Return to step 3         13x for 1ng cDNA   inputs  11x for 5ng cDNA inputs       7  72  00:05:00      8  4  hold    
 
  
SECTION: D: Ampure bead purification 2
9. 
 
Make sure beads are equilibrated to Room temperature.

SECTION: D: Ampure bead purification 2
10. Add 32.5 µL-35 µL Ampure beads/sample (0.65x-0.7x beads:sample ratio), seal, mix, brief spin.

SECTION: D: Ampure bead purification 2
11. Incubate sample/bead mix @Room temperature for 5 min.

SECTION: D: Ampure bead purification 2
12. Move to magnet stand, allow solution to clear, discard supernatant.
SECTION: D: Ampure bead purification 2
13. Wash 1 with 200 µL of 80% EtOH.

SECTION: D: Ampure bead purification 2
14. Wash 2 with 200 µL of 80% EtOH.

SECTION: D: Ampure bead purification 2
15. Elute in 20 µL water. Add water to samples, take off magnet, seal, mix, spin, incubate @Room temperature for 2 min.

SECTION: D: Ampure bead purification 2
16. Transfer samples back to magnet stand, allow to clear, save supernatant in separate, clean tubes.
SECTION: D: Ampure bead purification 2
17. REPEAT Ampure bead purification 2 for a total of 2x bead cleanups.
SECTION: D: Ampure bead purification 2
18. Evaluate on Tapestation.