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Protocols
NextSeq 2000

NextSeq 2000

CHMI owns and operates its own NextSeq 2000. This instrument is ideal for a wide range of experiments, including RNA-seq, scRNA-seq, genome sequencing, and much more.

Reference material

Thawing the Reagent Cartridge

Option 1 - Thawing in a water bath

Take the reagent cartridge out of the box but leave it in the foil bag
Prepare a 25°C water bath of 9.5-10 cm depth
Place the reagent cartridge in the water (it will float) with the label face up
Leave the reagent cartridge in the 25°C water bath for 6-8 hours (do not exceed 8 hours)
Remove the cartridge from the water bath after 6-8 hours and pat dry with paper towels

Option 2 - Thawing in the refrigerator

One day prior to your sequencing run, take the reagent cartridge out of the box but leave it in the foil bag
Place the the cartridge on the bench with the label face up so air can circulate on all times
Leave the reagent cartridge on the bench for 6 hours
Place the cartridge in a 2-8°C refrigerator with the label face up so air can circulate on all times
Leave the reagent cartridge in the refrigerator for 12-72 hours (do not exceed 72 hours)

Option 3 - Thawing on the bench

One day prior to your sequencing run, take the reagent cartridge out of the box but leave it in the foil bag
Place the the cartridge on the bench with the label face up so air can circulate on all times
Leave the reagent cartridge on the bench for 9-16 hours (do not exceed 16 hours)

Pooling

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. 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.
Thaw Illumina’s RSB with Tween for use in the pool.
Normalize and pool all your libraries to 2 nM in a LoBind microcentrifuge tube, using the RSB with Tween.
Quantify your pool on Qubit and Tapestation and enter into the calculator sheet to check that your pool is close to the 2 nM concentration.

Final Dilution

Make sure your flow cell and reagent cartridge are out at room temperature (15 minutes to an hour). In addition to these sequencing reagents, you will need your 2 nM pool, 1 nM PhiX (in the -20°C refrigerator) and more RSB with Tween.
Get a new 1.5 mL LoBind tube.
Using the table below, dilute your 2 nM pool to a final volume of 24 µL:

Final Library Concentration

Library TypeLoading Conc. (pM)2 nM Library Volume (µL)RSB with Tween 20 Volume (µL)
10X Chromium Next GEM Single Cell Multiome ATAC
650
7.8
16.2
10X Chromium Next GEM Single Cell ATAC
Takara Pico v3 Kit
1000
12
12
Takara SMART Seq HT PLUS Kit
1000
12
12
10X Chromium Next GEM 3’ Gene Expression Kit v3.1 (also for Multiome Gene Expression)
650
7.8
16.2
Illumina Stranded mRNA Prep
750
9
15
TruSeq DNA Nano 550
1500
18
6
TruSeq Stranded mRNA
1000
12
12
100% PhiX
650
7.8
16.2
Illumina DNA PCR-Free
1000
12
12
TruSeq DNA Nano 350
1200
14.4
9.6
Illumina DNA Prep
750
9
15
Illumina DNA Prep with Enrichment
1000
12
12
Illumina Stranded Total RNA with Ribo-Zero Plus
750
9
15
Ampliseq for Illumina Library PLUS
750
9
15
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For unlisted library types, start with a loading concentration of 650 pM and optimize over subsequent runs
If adding PhiX, use an aliquot of 1 nM PhiX:
Add 1 µL 1 nM PhiX to the 24 µL library for a total of 25 µL (this is a ~2% PhiX spike-in)
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If starting with the 10 nM stock PhiX: Prepare 20 µL 1 nM PhiX by combining 2 µL 10 nM PhiX and 18 µL RSB
Vortex final library briefly and centrifuge at 280 xG for 1 minute
Set aside on ice

Load Consumables

Make sure you have your flow cell and reagent cartridge out at room temperature, as well as your final library on ice
Open the cartridge bag by tearing or cutting with scissors from the top notch on either side
Remove the cartridge from the bag
Invert the cartridge 10 times to mix reagents
  • Invert side-to-side, not front-to-back
  • Internal components can rattle during inversion, which is normal
    • image
Open the flow cell package by tearing or cutting with scissors and pull the flow cell out of the package
Hold the flow cell by the grey tab with the label on the tab facing up
Push the flow cell into the reagent cartridge until there is an audible click. When properly loaded, the grey tab protrudes from the cartridge.
Carefully pull back the grey tab to expose the flow cell. Recycle the tab.
Do Not wipe the surface of the flow cell with ethanol
Using a new P1000 pipette tip, pierce the library reservoir and push the foil to the edges to enlarge the hole
Discard the pipette tip to prevent contamination
Add 20 µL diluted library to the bottom of the reservoir by slowly lowering the pipette tip to the bottom of the reservoir before dispensing.
  • Avoid touching the foil
  • You will not be able to see the bottom of the well, so you will have to feel the bottom of the well
Bring your loaded cartridge to the sequencer
Press “Start” and follow the on-screen prompts
The first round of system checks takes about 8 minutes
The second round of system checks (checking fluidics) takes about 6 minutes

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