Thawing and reagent preparation guidelines for the NextSeq 1000/2000 reagents

There are three validated methods for thawing a NextSeq 1000/2000 reagent cartridge.

1. Controlled Water Bath

2. Refrigerator

3. Room Temperature

Table 1: Summary of Thaw Methods for 100-cycle, 200-cycle, or 300-cycle kits

Method	Minimum Time to Complete Thaw	Do Not Exceed	Storage of Thawed Cartridge*
25°C Controlled Water Bath	6 hours	8 hours	72 hours at 2-8°C
Refrigerator	6 hours at Room Temp + 12 hours at 2-8°C	72 hours at 2-8°C	60 hours at 2-8°C
Room Temperature	9 hours	16 hours	72 hours at 2-8°C

*after minimum thaw time

Table 2: Summary of Thaw Methods for 600-cycle kits

Method	Minimum Time to Complete Thaw	Do Not Exceed	Storage of Thawed Cartridge*
25°C Controlled Water Bath	8 hours	10 hours	72 hours at 2-8°C
Refrigerator	6 hours at Room Temp + 16 hours at 2-8°C	72 hours at 2-8°C	56 hours at 2-8°C
Room Temperature	12 hours	16 hours	72 hours at 2-8°C

Table 3: Summary of Thaw Methods for XLEAP kits

Method	Minimum Time to Complete Thaw	Do Not Exceed	Storage of Thawed Cartridge*
25°C Controlled Water Bath	8 hours	10 hours	72 hours at 2-8°C
Refrigerator	6 hours at Room Temp + 16 hours at 2-8°C	72 hours at 2-8°C	56 hours at 2-8°C
Room Temperature	12 hours	16 hours	72 hours at 2-8°C

The NextSeq 1000/2000 system integrates many of the fluidics components into the reagent cartridge. As a result, the cartridge will rattle even if the cartridge has been thawed correctly. There is no way to visually or audibly confirm the cartridge has been thawed. Thaw according to these validated guidelines.

Note: All thawing methods require thaw in an unopened foil bag with the label facing up.

Controlled Water Bath: *Best for thawing and start run the same day.*1. Put on a new pair of powder-free gloves and remove the cartridge from storage. 2. Remove the cartridge from the box but do not open the silver foil bag.

• Thawing a torn or punctured bag in a water bath can result in sequencing failure. If the bag is punctured, thaw via refrigerator or room temperature method instead.

1. Thaw the bagged cartridge in a controlled 25ºC water bath as follows.

• For P1, P2, or P3 Reagent Kits (100-cycle, 200-cycle, 300-cycle), thaw for 6 hours. Do not exceed 8 hours in water batch

• For P1 or P2 Reagent kits (600-cycle), thaw for 8 hours. Do not exceed 10 hours in water bath.

• For XLEAP Kits (P1-P4, 100/200/300/600-cycle), thaw for 8 hours. Do not exceed 10 hours in water bath.

• Maintain 9.5 - 10 cm minimum water depth regardless of how many cartridges are thawing.

• Set a temperature-controlled water bath of 25ºC.

• Face the bag label up and set it in a water bath without submerging.

  • Do not weigh the cartridge down to submerge.

• Do not simultaneously thaw more cartridges than supported by the water bath.

• Do not stack cartridges.

• Water bath recommendations can be found in the Ancillary Equipment section of the NextSeq 1000/2000 Product Documentation.

1. Remove the cartridge from the water bath and dry with paper towels.

Refrigerator: Best for next morning run start or weekend thaw.

1. Put on a new pair of powder-free gloves.

2. One day prior to the anticipated run, remove the cartridge from -25ºC to -15ºC storage.

3. Remove the cartridge from the box but do not open the silver foil bag.

4. Position the cartridge at room temperature so that the label faces up and air can circulate on the sides and top.

• If the bag label is not facing up, sequencing data will be negatively impacted.

1. Thaw at room temperature for 6 hours, with air on all sides.

2. Position cartridge in a 2ºC - 8ºC refrigerator so that the label faces up and air can circulate on sides.

3. Thaw in refrigerator as follows:

• For P1, P2, or P3 Reagents Kits (100-cycle, 200-cycle, or 300-cycle), thaw for 12 hours. Do not exceed 72 hours.

• For P1 or P2 reagents (600-cycle), thaw for 16 hours. Do not exceed 72 hours.

• For XLEAP reagents (P1-P4, 100/200/300/600-cycle), thaw for 16 hours. Do not exceed 72 hours.

Begin thawing at least 1 day before starting the run.

Room Temperature:

1. Put on a new pair of powder-free gloves.

2. Remove the cartridge from -25ºC to -15ºC storage.

3. Remove cartridge from the box but do not open the silver foil bag.

4. Position the cartridge so that the label faces up and air can circulate on the sides and top.

• If the bag label is not facing up, sequencing data will be negatively impacted.

1. Thaw at room temp as follows

• For P1, P2, or P3 Reagents kits (100-cycle, 200-cycle, or 300-cycle), thaw for 9 hours. Do not exceed 16 hours.

• For P1 or P2 Reagents kits (600-cycle), thaw for 12 hours. Do not exceed 16 hours.

• For XLEAP Reagents (P1-P4, 100/200/300/600-cycle), thaw for 12 hours. Do not exceed 16 hours.

All Methods: After completing the cartridge thaw, the cartridge can be stored at 4°C for up to 72 hours total. If stored at 4°C, the cartridge must be brought to room temperature for 15 minutes to 1 hour prior to starting the sequencing run.

• For 100-cycle, 200-cycle, or 300-cycle cartridges prepared via refrigerator method do not exceed 60 hours. For 600-cycle cartridges prepared via refrigerator method, do not exceed 56 hours.

• For XLEAP cartridges prepared via refrigerator method, do not exceed 56 hours.

Prepare the flow cell and cartridge

1. Prepare the flow cell as follows.

• Remove a new flow cell from 2-8ºC storage.

• Set the unopened package at room temperature for 10-15 minutes to prevent condensation when removing the flow cell from the package. Preparing the flow cell now ensures that it reaches room temperature on time to start the run.

1. Prepare the Cartridge

• If using the refrigerator thaw method or removing it from fridge storage, remove the thawed cartridge from 2-8ºC storage. Set the unopened cartridge aside at room temperature for at least 15 minutes before sequencing. Do not exceed 1 hour.

• If using room temperature thaw, proceed to Load Consumables Step

Load consumables into the cartridge Prepare the Cartridge1. Open the cartridge bag by tearing or cutting with scissors from the top-notch on either side. 2. Remove the cartridge from the bag. Discard the bag and desiccant. 3. Invert the cartridge 10 times to mix reagents. Internal components can rattle during inversion, which is normal. ****

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1. FOR XLEAP CONSUMABLES ONLY

• • Lift and tap cartridge FIVE times. - Please see linked video for guidance: NextSeq 1000/2000 XLEAP SBS Cartridge Preparation Video

Load the Flow Cell

1. Open the silver foil package by tearing or cutting with scissors from the top slit on either side. If unable to use the flow cell immediately, return to storage.

2. Pull the flow cell out of the package. Set aside the foil package and desiccant in case the flow cell needs to be returned to storage. The desiccant is contained in a pouch at the bottom of the foil package. Discard them when sequencing begins.

1. Hold the flow cell by the gray tab with the label facing up.

2. Push to insert the flow cell into the slot on the front of the cartridge. An audible click indicates that the flow cell is in place. When properly loaded, the gray tab protrudes from the cartridge.

1. Pull back and remove the gray tab to expose the flow cell. Recycle the tab.

If unable to start run after opening consumables, return the flow cell to original foil packaging with desiccant, tape the packaging shut and store at 4ºC for up to 72 hours.

If foil package has not been opened flow cell can be placed back at 4ºC (no limit on the number of times brought to room temp).

For more information on storing opened reagents, search Knowledge Base Article Returning NextSeq 1000/2000 reagents to storage

For more information, including on library loading, see the NextSeq 1000/2000 Product Documentation

For any feedback or questions regarding this article (Illumina Knowledge Article #2430), contact Illumina Technical Support [e-mail].