TITLE: Fixed RNA - GEM Recovery - Sequencing 
AUTHORS: Ksenija Sabic

[DESCRIPTION]
This protocol is a continuation of CG000527 (Step 3 and onward).

[BEFORE_START]
Prepare daily fresh 80% Ethanol for wash steps.

[GUIDELINES]
Please review and consult the full 10x Genomics protocols prior to starting and at any point during the procedure if needed.  

[STEPS]
SECTION: GEM Recovery and Pre-Amplification
2. Add 125 µL Recovery Agent to each sample at room temperature. 

DO NOT pipette mix or vortex the biphasic mixture.
SECTION: GEM Recovery and Pre-Amplification
3. Firmly secure the cap on the tube strip, ensuring that no liquid is trapped between the cap and the tube rim. Mix by inverting the capped tube strip 5x.

DO NOT invert without firmly securing the caps.
SECTION: GEM Recovery and Pre-Amplification
4. Wait 2 min.

The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (translucent/opaque). A smaller aqueous phase volume indicates a clog during GEM generation.
SECTION: GEM Recovery and Pre-Amplification
5. Centrifuge briefly.
SECTION: GEM Recovery and Pre-Amplification
6. Slowly remove and discard 125 µL Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
SECTION: GEM Recovery and Pre-Amplification
7. Proceed directly to Pre-Amplification PCR. No cleanup step is required.
SECTION: GEM Recovery and Pre-Amplification
8. Prepare Pre-Amplification Mix on ice. Vortex and centrifuge briefly.
 

SECTION: GEM Recovery and Pre-Amplification
9. Add 35 µL Pre-Amplification Mix to aqueous sample from step 6.

SECTION: GEM Recovery and Pre-Amplification
10. Cap firmly and invert 8x to mix. Centrifuge briefly.
SECTION: GEM Recovery and Pre-Amplification
11. Incubate in a thermal cycler with the following protocol:
 
Store at 4°C for up to 72 h or −20°C for ≤1 week, or proceed to the next step.
SECTION: GEM Recovery and Pre-Amplification
12. Prepare Elution Solution. Vortex and centrifuge briefly.
 

SECTION: GEM Recovery and Pre-Amplification
13. Centrifuge the sample (PCR product) for 30 s sec in a microcentrifuge and transfer 70 µL of the upper layer to a new tube.

Presence of a cloudy precipitate at the interface between phases is normal. Avoid transferring the precipitate when transferring 70 μl at this step.
SECTION: GEM Recovery and Pre-Amplification
14. Vortex to resuspend the SPRIselect reagent. Add 126 µL SPRIselect reagent (1.8X) to each sample and pipette mix 15x (pipette set to 180 μl).
SECTION: GEM Recovery and Pre-Amplification
15. Incubate 5 min min at Room temperature.

SECTION: GEM Recovery and Pre-Amplification
16. Place on the magnet (high position) until the solution clears.
SECTION: GEM Recovery and Pre-Amplification
17. Remove the supernatant. DO NOT discard any beads.
SECTION: GEM Recovery and Pre-Amplification
18. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet. Wait 30 s sec.
SECTION: GEM Recovery and Pre-Amplification
18.1. Remove the ethanol.
SECTION: GEM Recovery and Pre-Amplification
18.2. Repeat steps 18 and 18.1 for a total of 2 washes.
SECTION: GEM Recovery and Pre-Amplification
19. Centrifuge briefly and place on the magnet (low position).
SECTION: GEM Recovery and Pre-Amplification
20. Remove any remaining ethanol. DO NOT let the sample dry to ensure maximum elution efficiency.
SECTION: GEM Recovery and Pre-Amplification
21. Remove from the magnet. Add 101 µL Elution Solution (from step 12). Wait 1 min min before resuspending. Pipette mix 15x.

SECTION: GEM Recovery and Pre-Amplification
22. Incubate 2 min min at Room temperature.

SECTION: GEM Recovery and Pre-Amplification
23. Place the tube strip on the magnet (high position) until the solution clears.
SECTION: GEM Recovery and Pre-Amplification
24. Transfer 100 µL sample to a new tube strip.
SECTION: GEM Recovery and Pre-Amplification
25. Store at 4 °C for ≤4320 min hours or at -20 °C for ≤4 weeks, or proceed to the next step.
SECTION: Library Construction
26. Reagent Preparation (~10 min):
SECTION: GEM Recovery and Pre-Amplification
1. Reagent Preparation (~15 mins):
SECTION: GEM Recovery and Pre-Amplification
1.1. Thaw Reducing Agent B at Room temperature, vortex, verify no precipitate, centrifuge briefly.
SECTION: GEM Recovery and Pre-Amplification
1.2. Thaw Pre-Amp Primers B at  Room temperature, vortex, centrifuge briefly.
SECTION: GEM Recovery and Pre-Amplification
1.3. Keep Amp Mix  on ice. Vortex and centrifuge briefly.
SECTION: GEM Recovery and Pre-Amplification
1.4. Prepare fresh 80% Ethanol (2.5 mL for 4 GEM reactions).
SECTION: Library Construction
26.1. Thaw Dual Index Plate TS Set A at Room temperature.
SECTION: Library Construction
26.2. Keep Amp Mix  on ice. Vortex and centrifuge briefly.
SECTION: Library Construction
26.3. If planning to assess quality and concentration of library immediately after completion, thaw Agilent Bioanalyzer High Sensitivity Kit at Room temperature, keeping prepared gel and gel-dye away from light sources. Consult the following protocol for details:
 

SECTION: Library Construction
27. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x sample index name (PN-3000511 Dual Index Plate TS Set A well ID) used.
SECTION: Library Construction
28. Prepare Sample Index PCR Mix on ice:
 

SECTION: Library Construction
29. Transfer ONLY 20 µL of sample from the step 24 to a new tube strip.
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30. Add 60 µL Sample Index PCR Mix to 20 μl sample.
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31. Add 20 µLof an individual Dual Index TS Set A to each sample. Pipette mix 5x (pipette set to 90 μl). Centrifuge briefly.
SECTION: Library Construction
32. Incubate in a thermal cycler with the following protocol:
 

SECTION: Library Construction
32.1. 
 

SECTION: Library Construction
33. Store at 4 °C for ≤72 h, or proceed to the next step.
SECTION: Library Construction
34. Vortex to resuspend the SPRIselect reagent. Add 100 µL SPRIselect Reagent (1.0X) to each sample. Pipette mix 15x (pipette set to 180 μl).
SECTION: Library Construction
35. Incubate 5 min min at Room temperature.

SECTION: Library Construction
36. Place on the magnet (high position) until the solution clears.
SECTION: Library Construction
37. Remove the supernatant. DO NOT discard any beads.
SECTION: Library Construction
38. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet. Wait 30 s sec.
SECTION: Library Construction
38.1. Remove the ethanol.
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38.2. Repeat steps 38 and 38.1 for a total of 2 washes.
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39. Centrifuge briefly and place on the magnet (low position).
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40. Remove any remaining ethanol. DO NOT let the sample dry to ensure maximum elution efficiency.
SECTION: Library Construction
41. Remove from the magnet. Add 41 µL Buffer EB. Pipette mix 15x.

SECTION: Library Construction
42. Incubate 2 min min at Room temperature.

SECTION: Library Construction
43. Place on the magnet (low position) until the solution clears.
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44. Transfer 40 µL to a new labeled DNA LoBind tube.

SECTION: Library Construction
45. Store at 4 °C for up to 72 h, -20 °C or at -80 °C for long-term storage.

SECTION: Library Construction
46. Run 1 µL sample at 1:80 dilution on an Agilent Bioanalyzer High Sensitivity chip. Select the region between 150-300 bp to determine average size of the library. Consult the following protocol:
 



SECTION: Sequencing
47. Consult the following protocol to send the libraries for sequencing:
 

SECTION: Library Construction
46.1. See image below for representative trace:

 

SECTION: Library Construction
44.1. Make a 1:80 dilution of the library for quantification.