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Capture area generation

The following section details the generation of capture areas for the Open-ST protocol.

By sequencing oligos, which comprise unique 32-nucleotide barcodes, appropriate adapters, and a poly-dT, we register the barcode sequences and their associated coordinates on the flow cell.

For instance, you can get ~360 capture areas sized 3x4 mm from a single Illumina® NovaSeq 6000 S4 flow cell.

Sequencing of barcoded library

When using an Illumina® NovaSeq 6000 S4 flow cell (35 cycles), sequence the HDMI32-DraI library (see in Oligonucleotides) at a loading concentration of 200 pM. Using 200 pM library, loaded according to the KAPA qPCR value, we obtained the following quality metrics for the barcoded fc_1 used in our our preprint: Q30 >= 86%; PF = 78%; occupied = 97%. Although great results were achieved using this flow cell, 97% occupied is high. Consequently, we suggest to use a titration of library loading concentrations (one concentration per lane) when generating your first barcoded flow cell.

Sequence a single-end 37 cycle read, using Read1-DraI oligo as a custom primer. Use a custom sequencing recipe that stops the run immediately after read 1 prior to on-instrument washes.


The custom recipe published in our bioarchive pre-print and linked above was provided by Illumina Technical Support. It was used in a sequencing run with the following versions: reagent kit v1.5, RTA v3.4.4, Flow Cell Consumable v1, Sbs Consumable v3, NovaSeq control Software v 1.7.5 (in the pre-print) and v 1.8.1 (in an independent flow cell with no published data). Be aware that the custom recipe may change with different versions.

Expected (data) output

Either when using your own sequencing equipment or relying on a sequencing facility, you will get access to (most likely) already demultiplexed fastq files; otherwise, you can get access to raw basecall files in bcl format.

Either of these files shall be used as the input for openst later, to create a database of barcode sequences and their spatial locations.

Enzymatic processing


  • Prepare mixes in excess. ~300 μL per lane is required for the S4 flow cell.
  • If bubbles occur, mark these with pen on the flow cell. Repeat reactions if many bubbles occur and ensure bubbles do not form at the same locations.
  • Use a P1000 pipette and pipette slowly to avoid the formation of bubbles.
  • For removing washes, pipette the liquid out and then blow through air using the P1000 to remove remaining liquid.

Dra I digestion

DraI mix

Reagent Final concentration Volume (μL)
DraI 2U/μL 10
10X CutSmart buffer 1x 10
Ultrapure water 80
  1. Wash flow cell by flowing through 500 μL ultrapure water using a P1000 pipette.
  2. Add DraI mix and incubate at 37°C overnight.

Exonucelase I digestion

ExoI mix

Reagent Final concentration Volume (μL)
ExoI 1 U/μL 5
10X ExoI buffer 1x 10
Ultrapure water 85
  1. Wash flow cell by flowing through 500 μL 80% ethanol, then ultrapure water.
  2. Add Exonuclease I mix and incubate for 45 min at 37°C.
  3. Wash flow cell by flowing through 500 μL ultrapure water three times.

Opening, denaturation and washes


The NovaSeq6000 S4 flow cell images the top and bottom glass. Thus, keep both and take care not to break them.


Check out our video linked at the end of this page to see a demonstration of the opening of the flow cell.

  1. Remove the flow cell from its plastic encasing.
  2. Carefully score along the sides of the flow cell using a scalpell. The blade should be in one plane with the flow cell.
  3. Once all sides detach, carefully seperate the two flow cell glasses.
  4. Denature the second strand by washing the opened flow cell surfaces in a beaker of 0.1N NaOH, incubating for 5 min.
  5. After the denaturation, wash the surfaces 3x with 0.1M Tris-HCl (pH 7.5) and then 3x with Ultrapure water.
  6. Let the flow cell surfaces air dry before proceeding with the scoring and breaking of the flow cell surfaces into smaller capture areas.

Breaking the flow cells into capture areas


Capture areas can be stored dry at -20°C for extended periods of time. We have generated libraries from prepared capture areas stored for 12 months.

We have designed a cutting guide that facilitates the breaking of the flow cell into regular capture areas. We provide the 3D model of the cutting guide as a printable stl file. If you don't have a 3D printer, you can check for 3D printing services near you - they will help you in this endeavor 🤗.