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Protocols

NextSeq 500 (Legacy)

CHMI owns and operates its own NextSeq 500. This instrument is ideal for a wide range of experiments, including RNA-seq, scRNA-seq, genome sequencing, and much more.

Reference material

Pooling

If you plan to sequence on the same day, place the flow cell at room temperature and put the reagent cartridge in a water bath to thaw before starting your pool. The flow cell needs to equilibrate for at least half an hour and the reagent cartridge needs a similar amount of time to thaw (depending on kit, sometimes more time). You can also thaw the reagent cartridge in the fridge or cold room overnight. Once thawed, use within a week.

Quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library.
Run your samples on Tapestation with either the D1000, HSD1000, or HSD5000 assay, depending on library prep kit. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
Download our nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. Normalize and pool all your libraries to 4, 2, 1, or 0.5 nM in a LoBind microcentrifuge tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
Quantify your pool on Qubit and enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.

Denaturation and Dilution

The following protocol is based on the NextSeq Denature and Dilute Guide (linked above), with few adjustments. The biggest change is that we do not vortex the libraries after denaturation.

Before proceeding to these steps, make sure your flow cell is out at room temperature and your reagent cartridge is thawing. In addition to these sequencing reagents, you will need pooled libraries, 20pM denatured PhiX, 1M Tris-HCl ph 8, 1N NaOH, MilliQ water, and HT1 buffer.

  1. Get 3 1.5mL tubes and 1 2mL tube.
  2. In one of the 1.5mL tubes, make a 0.2N dilution of NaOH by adding 800uL of MilliQ water and 200uL 1N NaOH. Vortex and set aside.
  3. In one of the 1.5mL tubes, make a 200mM dilution of Tris by adding 800uL of MilliQ water and 200uL 1M Tris. Vortex and set aside.
  4. Label the third 1.5mL tube "20pM." In this tube, add the volumes of NaOH and library pool in the following chart, according to pool nM concentration:

Denaturation

Library Pool nM Concentrationul 0.2 N NaOHul Library Pool
4
5
5
2
10
10
1
20
20
0.5
40
40

5. Pipette to mix.

6. Incubate at room temperature for 5 minutes.

7. Immediately add the appropriate volume of 200mM Tris and pipette to mix:

Tris quench

Library Pool nM Concentrationul 200mM Tris
4
5
2
10
1
20
0.5
40

8. Add the appropriate volume of HT1:

Ht1

Library Pool nM Concentrationul prechilled HT1
4
985
2
970
1
940
0.5
880

9. Slowly pipette to mix or invert the tube. Your "20pM" tube now contains your denatured library pool at 20pM concentration.

10. Proceed to the final dilution of your samples. Label your 2mL tube "Final" and add the appropriate volumes of HT1, 20pM library, and 20pM PhiX:

NextSeq Final Dilution

Desired Loading Concentration pMul HT1ul 20pM Library Poolul 20pM PhiX for 1% Spike-Inul 20pM PhiX for 5% Spike-Inul 20pM PhiX for 20% Spike-In
1.3
1215.5
84.5
0.85
4.23
16.90
1.4
1209.0
91.0
0.91
4.55
18.20
1.5
1202.5
97.5
0.98
4.88
19.50
1.6
1196.0
104.0
1.04
5.20
20.80
1.7
1189.5
110.5
1.11
5.53
22.10
1.8
1183.0
117.0
1.17
5.85
23.40

11. Invert to mix and keep on ice until ready to use.

Loading the Sequencer

1. Make sure the flow cell has been at room temperature for at least 30 minutes to equilibrate. Bring it to the sequencer and tear open the foil at the perforation on the shorter end.

2. Open the plastic clamshell and carefully remove the flow cell. It is snugly wedged in, so slowly pull it at an angle until it releases.

3. Hold the flow cell to the light and inspect the lanes for any scratches or defects. Inspect the back and make sure the gaskets are seated properly.

4. Put some ethanol on a kim wipe, and slowly wipe the glass face of the flow cell in a single direction. Take a clean kim wipe and repeat to remove any excess ethanol or remaining dust.

5. Press "Sequence" and log in to Basespace.

6. Following the on-screen prompts, load the flow cell and select Next. The pins on the machine line up with the holes on the flow cell.

7. Remove the Buffer Cartridge and open up the foil holes with gloves fingers. This waste can be disposed down the sink and, once empty, the cartridge can be put in the regular trash.

8. New Buffer Cartridges are kept in the cabinet below the sequencer. These are interchangeable between all NextSeq v2.5 kits. Open a new one and slide into position.

9. Remove the Spent Reagents Container. There is a chemical waste carboy around the corner from the sequencer. Open the container by sliding open the grey circular opening and empty it in to the carboy completely. Close the container and wipe off excess before placing it back in the sequencer. This waste contains formamide. Never dispose it down the sink and change gloves if you spill or drip.

10. Remove the old Reagent Cartridge from the sequencer. Remove the reservoir behind position #6. This contains trace amounts of formamide and is disposed in a bag for chemical waste pickup. The rest of the cartridge can be disposed in regular trash.

11. Return to your new, thawed reagent cartridge. Gently tilt it back and forth to check thaw and mix reagents.

12. Hold a p1000 tip in your hand and stab into the foil of well #10 on the reagent cartridge. Open up the foil of the well completely.

13. Pipette your denatured, diluted pool into the well. Pipette the entire contents of the well, including any that got into the cap. You will have about ~1300ul total.

14. Load the reagent cartridge into the sequencer.

15. Follow the on-screen prompts, selecting your Basespace run and confirming the parameters.

16. After the self-check, press Start!