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sequencing
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scg-protocols-v1

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## Example: Library Construction Input Mass & SI PCR Cycles (continued)
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## 3.1 GEX Fragmentation, End Repair &amp; A-tailing
- a. Prepare a thermal cycler with the following incubation protocol.
- b. Vortex Fragmentation Buffer. Verify there is no precipitate.
- c. Prepare Fragmentation Mix on ice. Add reagents in the order listed. Pipette mix and centrifuge briefly.
- d. Transfer ONLY 10 μl purified cDNA sample from Cleanup (step 2.3) to a tube strip. Note that only 10 μl (25%) cDNA sample is sufficient for generating Gene Expression library. The remaining cDNA sample can be
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| Lid Temperature | Reaction Volume | Run Time |
|---------------------------------------------------------|-------------------|---------------|
| 65°C | 50 μ l | ~35 min |
| Step | Temperature | Time hh:mm:ss |
| Pre-cool block | | |
| Pre-cool block prior to preparing the Fragmentation Mix | 4°C | Hold |
| Fragmentation | 32°C | 00:05:00 |
| End Repair & A-Tailing | 65°C | 00:30:00 |
| Hold | 4°C | Hold |
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| Fragmentation Mix Add reagents in the order listed | PN | 1X ( μ l) | 4X + 10% ( μ l) | 8X + 10% ( μ l) |
|------------------------------------------------------|------------------|-------------|-------------------|-------------------|
| Buffer EB | - | 25 | 110 | 220 |
| Fragmentation Buffer | 2000091 | 5 | 22 | 44 |
| Fragmentation Enzyme | 2000090/ 2000104 | 10 | 44 | 88 |
| Total | | 40 | 176 | 352 |
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- stored at 4°C for up to 72 h or at -20°C for up to 4 weeks for generating additional Gene Expression libraries.
- e. Add 40 μl Fragmentation Mix to each 10 μl sample.
- f. Pipette mix 15x (pipette set to 30 μl) on ice. Centrifuge briefly.
- g. Transfer into the pre-cooled thermal cycler ( 4°C ) and press 'SKIP' to initiate the protocol.
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## 3.2 GEX Post Fragmentation, End Repair &amp; A-tailing Double Sided - SPRIselect
- a. Vortex to resuspend SPRIselect reagent. Add 30 μl SPRIselect reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 75 μl).
- b. Incubate 5 min at room temperature .
- c. Place on the magnet· High until the solution clears. DO NOT discard supernatant.
- d. Transfer 75 μl supernatant to a new tube strip.
- e. Vortex to resuspend SPRIselect reagent. Add 10 μl SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 80 μl).
- f. Incubate 5 min at room temperature .
- g. Place on the magnet· High until the solution clears.
- h. Remove 80 μl supernatant. DO NOT discard any beads.
- i. With the tube strip still on the magnet, add 125 μl 80% ethanol to the pellet. Wait 30 sec .
- j. Remove the ethanol.
- k. Repeat steps i and j for a total of 2 washes.
- l. Centrifuge briefly. Place on the magnet· Low until the solution clears.
- m. Remove remaining ethanol. DO NOT over dry to ensure maximum elution efficiency.
- n. Remove from the magnet. Add 50.5 μl Buffer EB to each sample. Pipette mix 15x (pipette set to 45 µ l).
- o. Incubate 2 min at room temperature .
- p. Place on the magnet· High until the solution clears.
- q. Transfer 50 μl sample to a new tube strip.
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