TITLE: Loading a Radseq Library on Nextseq 500
AUTHORS: Angie Chia

[DESCRIPTION]
Protocol to load a Radseq Library on EFGL Nextseq 500

[STEPS]
SECTION: NextSeq 500 Prep
2. Thaw Reagent Tray and Flow Cell - It is important that the output of both the reagent tray and flow cell matches i.e. MID/MID or HIGH/HIGH
SECTION: NextSeq 500 Prep
2.1. Retrieve a High-300 output or Mid-300 output Illumina Reagent Tray from the freezer and place in bucket/container of room temperature tap water. Water level should be to the bottom of the reagent tray handle. Let thaw for 60 min in water bath.

SECTION: NextSeq 500 Prep
2.2. Retrieve 1 tube of HT1 buffer from the freezer, and place in a beaker of room temperature tap water to thaw as well. 
SECTION: NextSeq 500 Prep
2.3. At the 30 minute mark of reagent tray thaw process, retrieve a matching output flow cell (i.e. MID/MID or HIGH/HIGH) from the fridge. Open flow cell packaging, but leave it in its packaging on bench to let it come to room temperature. 
SECTION: NextSeq 500 Prep
1. Power cycling & Manual Wash: Perform a manual wash (Post Run Manual Wash), approximately two hours prior to run/or on the morning of run. 
 

SECTION: NextSeq 500 Prep
1.1. Power cycle NextSeq 500 
Tap on Home, if not already on Home screen
Select Manage Instrument -> System Shut down Options
Select Shut Down
Wait 5 min, before powering back up by pressing on the On button in front of machine
Enter login credentials once Windows startup has completed.
Wait for machine to initialize NextSeq software, and displays Home screen once ready.

SECTION: NextSeq 500 Prep
1.2. Perform a Post Run Manual Wash (if a run was completed within the past 2-3 days, OK with quick wash instead)
On Home screen, select Perform Wash -> Manual Wash
Follow the instructions on NextSeq 500's screen 
When loading wash buffer tray, be sure to fill position 34 with 0.01% ddH20/TWEEN about 3/4 full 
Tap on Start to begin wash

Important: If there is a run in place prior to your wash, the waste container has 1.8% formamide. It needs to be disposed of properly into a waste bottle. Similarly, if reagent tray is in the machine, remove formamide container (location 6) and dispose of it properly (zip-loc bag - contaminated waste trash can). All other liquid/solid waste can be disposed of in sink/trash can. 

  
  

SECTION: NextSeq Library Prep
3. Begin prepping RAD library for loading, only after reagent tray has been thawing for 40 minutes, and flow cell left on bench for 10 minutes. 
SECTION: NextSeq Library Prep
3.1. Prepare 0.2 N NaOH denature solution. In a new 1.5mL LoBind tube (label tube 0.2):
Add 10 µL of 1 N NaOH + 40 µL of molecular grade lab water
Gently vortex, and briefly spin down.
Let sit at room temperature.

  
  

SECTION: NextSeq Library Prep
3.2. Add the following components in order into a new 1.5mL LoBind tube (label tube DL):
5 µL of 0.2 N NaOH + 5 µL of 4nM RAD library - do not pump, as this can introduce bubbles
Vortex gently, spin   (using IEC micromax)
Incubate DL at room temperature for 5 min
Add 5 µL of 200mM Tris-HCl pH 7.0 to DL
Vortex gently and spin down

  

SECTION: NextSeq Library Prep
3.3. To DL tube, add 985 µL of HT1 buffer. 

If library is not at 4nM, use the equation below to determine the amount of HT1 buffer to add:

 
SECTION: NextSeq Library Prep
3.4. Gently vortex DL tube, and spin down. Keep tube in a benchtop cooler. 

Library is now at 20pM. 
SECTION: NextSeq Library Prep
4. Depending on the output you'll want to achieve for your library, further dilute your library to the desired concentration prior to loading onto reagent tray for sequencing. The table below is an example that our lab uses for Mid/High - 300 cycles output libraries.
 
SECTION: Prep NextSeq reagent tray
5. Once reagent tray has been thawing for an hour, remove tray from water bath and wipe dry. 

Invert reagent tray gently 5 times to mix reagents, and gently tap tray on bench top to collect all liquid to the bottom.
Use a 1000µl pipette tip and punch a hole in position 10. Discard tip.
Load all 1300 µL of tube 10 into position 10 of reagent tray.

SECTION: Loading reagent tray on NextSeq 500
6. Verify both reagent tray and flow cell output matches. 
Take a new buffer cartridge
Inspect the flow cell by making sure brackets on the back are in place, and flushed. If there's any dirt on top of flow cell, gently remove with a Kimwipe. 
SECTION: Loading reagent tray on NextSeq 500
6.1. On Home screen, select Sequence
"Manual" would been pre-selected, tap on Next
SECTION: Loading reagent tray on NextSeq 500
6.2. Enter Run Parameters:
Run Name: Use your library name (e.g. L0012)
Library ID: Same as Run Name
Check the "Paired end" box
Read 1: 150    Index 1: 6    Index 2: 6    Read 2: 150    (index cycles are optional)
Tap on Next
SECTION: Loading reagent tray on NextSeq 500
6.3. Follow the instructions on NextSeq 500's screen to load buffer cartridge and reagent tray for sequencing.

Once all pre-run checks are complete on the machine, the run will start automatically. (Typical High-300 Output run takes ~32 hours, and Mid-300 Output takes ~26 hours)
SECTION: NextSeq Library Prep
4.1. In a new 2.0mL tube (label tube 10), add the components accordingly to the table above.
Gently vortex, and spin down. Tube 10 has a final volume of 1300 µL 
Keep tube in benchtop cooler till reagent tray is ready to be loaded.