TITLE: Nova-ST Chip Preparation Protocol
AUTHORS: Suresh Poovathingal, Kristofer Davie, Stein Aerts

[DESCRIPTION]
Nova-ST is a an open-source, high-resolution sequencing based spatial transcriptomics workflow. This method gives comparable resolution to BGI Stereoseq, SeqScope & PIXEL seq. Nova-ST is derived from dense nano-patterned randomly barcoded Illumina NovaSeq 6000 S4 sequencing flow cells. More details in the Nova-ST pre-print (https://www.biorxiv.org/content/10.1101/2024.02.22.581576v1). Nova-ST enables customized, low cost, flexible, and high-resolution spatial profiling of broad range of tissue section sizes (upto 10mm x 8 mm). In this protocol, we provide detailed step-by-step resource for implementing the Nova-ST spatial transcriptomics workflow in you lab. Bioinformatics and data analysis workflows are detailed in: https://github.com/aertslab/Nova-ST. For any protocol related or data analysis clarifications, you can reach out to us via nova.st.aertslab@gmail.com. 

[GUIDELINES]
Section: HDMI sequencing 

Oligonucleotide details:

HDMI32-DraI: CAAGCAGAAGACGGCATACGAGAT TCTTTCCCTACACGACGCTCTTCCGATCT NNVNBVNNVNNVNNVNNVNNVNNVNNVNNNNN TCTTGTGACTACAGCACCCTCGACTCTCGC TTTTTTTTTTTTTTTTTTTTTTTTTTT TTTAAA GACTTTCACCAGTCCATGAT GTGTAGATCTCGGTGGTCGCCGTATCATT

HDMI32-DraI was ordered using Ultramer service from IDT. Upon receipt, the HDMI-32 oligonucleotide were reconstituted in the Ultrapure water. The oligonucleotide was diluted to 10 nM before proceeding with the HDMI sequencing.

Read1-DraI: ATCATGGACTGGTGAAAGTC TTTAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA GCGAGAGTCGAGGGTGCTGTAGTCACAAGA

Read1-DraI was also ordered using from IDT with PAGE purification. Upon receipt, the Read1-DraI oligonucleotide was reconstituted in the Ultrapure water. The oligonucleotide was diluted to 100 nM before proceeding with the HDMI sequencing.

The HDMI32-DraI was diluted to 10 nM and the library and qPCR is used to estimate the library concentration. The concentration estimated from qPCR is used to normalize the HDMIDraI-32 library for the HDMI sequencing on Novaseq 6000 S4 flowcell.

Section: Preparation of Nova-ST chips

During the development of Nova-ST workflow we have developed two strategies for preparation of Nova-ST chips from the NovaSeq S4 flow cell and listed below are some consideration for choosing the appropriate method for cutting out the barcoded chips for Nova-ST workflow:

Manual cutting strategy: As described in our pre-print, this method is based on semi-automated glass cutting method, where we have used the NOMAD 3 CNC milling machine from Carbide3D to score the glass surface into a 1cm x 1cm cutting grid. This CNC milling machine comes with a 130W spindle and we used a diamond drag bit from the CNC milling machine manufacturer for scoring the glass surface. Post scoring, we used traditional glass running pliers (Speedwox, Amazon) to cut the glasses into 1 cm x 1 cm chip tiles. We also tried the manual cutting strategy proposed in Open-ST, another high resolution spatial transcriptomics workflow (https://www.biorxiv.org/content/10.1101/2023.12.22.572554v1). Both of these strategies, despite the numerous cutting optimizations and fine-tunings, we have found this procedure usually resulted in quite varied results, especially while cutting larger chip size of 1 cm2 tiles. Cutting of the thicker glass section is much more challenging to get precise cuts. In addition, the physical manipulation of the chips during the cutting process also introduces artifacts on the functionalized surface of the chip, which would directly affect the performance of the assay. Also, the first step of separating the think and thick glass layers is also quite challenging with uneven cuts and breakages. All-in-all, we find the manual glass cutting strategy to produce sub-optimal glass cuts and therefore resulting in reduced recovery of ST chips. In order to improve the chip recovery and to optimize the chip cutting strategy we adopted and moved to a more automated workflow.

Automated glass dicing strategy: In this new strategy, we use a wafer dicing instrument (DISCO DAD Automatic dicing saw; DAD 3220) to accurately cut the NovaSeq chips to varied dimensions. This instrument gives a great flexibility in cutting any desired dimensions. This instrument should commonly be available in electrical and electronics lab facilities. DAD 3220 employs a high-speed spindle (1.5 kW) fitted with an extremely thin diamond blade. The thickness of the cutting blades used varies with the material being cut, and in the case of glass cutting this is about  . The optimized glass dicing strategy provides close to 100% chip recovery. Manual intervention in the strategy is minimal and thus the artifact 

The protocol describes the details on both the cutting strategies. If access to the wafer dicing instrument is a possibility, its highly recommended to use this for cutting of Nova-ST chips due to the high reliability and reproducibility in getting accurately cut Nova-ST chips.

[STEPS]
SECTION: HDMI Sequencing
1. Before Starting
SECTION: HDMI Sequencing
1.1. The NovaSeq S4 reagents stored at -20 °C  is taken out and thawed at 4 °C  for atleast 960 min -1200 min  before sequencing. Prior to sequencing these reagents are removed and warmed to 25 °C  for atleast 30 min .

SECTION: HDMI Sequencing
1.2. The NovaSeq S4 flowcell stored at 4 °C is thawed to Room temperature at least 30 min before sequencing.

SECTION: HDMI Sequencing
1.3. Thaw HDMI32-DraI and Read1-DraI oligonucleotides to Room temperature, and prepare 20 µL of 100 micromolar (µM) of Read1-DraI read primer and prepare 200 µLof 1 micromolar (µM) of HDMI32-DraI oligonucleotide for HDMI sequencing. The  1 micromolar (µM) of HDMI32-DraI oligonucleotide is subject to qPCR quantification for library normalization

SECTION: HDMI Sequencing
2. Sequencing
SECTION: HDMI Sequencing
2.1. Prepare NaOH for denaturation (Mix the content well).

 

SECTION: HDMI Sequencing
2.2. Prepare Tris pH 8 (Mix the content well)
 

SECTION: HDMI Sequencing
2.3. Prepare Tris pH 8 for neutralization (Mix the content well)
 

SECTION: HDMI Sequencing
3. 1.5 nanomolar (nM)of normalized HDMI library is prepared by diluting the qPCR quantified HDMI32-DraI oligonucleotide using 10 mM Tris pH 8.0 buffer.
SECTION: HDMI Sequencing
4. Denaturing of the HDMI sequencing library is performed by adding 0.2 Molarity (M)NaOH to a final concentration of 0.04 Molarity (M).
SECTION: HDMI Sequencing
5. Incubate for 8 min. 
SECTION: HDMI Sequencing
6. Neutralize the denatured HDMI library by adding 0.4 Molarity (M) Tris pH 8 solution to a final concentration of 67 millimolar (mM). The final concentration of the HDMI library prior to loading on sequencer is 300 picomolar (pM) .
SECTION: HDMI Sequencing
7. Prepare the Read1-DraI primer concentration to 0.3 micromolar (µM) by adding 10.5 µL of 100 micromolar (µM)of Read1-DraI primer to 3489.5 µL of the HT1 buffer from the NovaSeq 6000 kit. Mix well and load the primer to the custom read primer 1 position in the NovaSeq reagent cartridge. 
SECTION: HDMI Sequencing
8. Proceed for HDMI sequencing. The sequencing configuration used for reading the HDMI barcodes is:
 

SECTION: HDMI Sequencing
9. At the end of 34 cycles, perform manual abortion of the run by pressing the option of "End run without Wash".
SECTION: HDMI Sequencing
10. The S4 flow cell is retrieved either for immediate downstream post processing steps, or it can also be stored safely at 4 °C  for at least 2 weeks (based on our experience). Users not having direct access to NovaSeq 6000 instrument can instruct sequencing facility to perform the HDMI sequencing and arrange for refrigerated transport of the flowcell in its original shipping case. In order the prevent the flow channels from evaporation, the inlet and outlet of the flow cell is plugged with 1.5 mm Polydimethylsiloxane (PDMS) punch.

Preparation of the 1.5 mm PDMS plugs:
SECTION: HDMI Sequencing
10.1. The monomer (part A) and catalyst (part B) of SYLGARD 184 Silicone Elastomer Kit is prepared in a 10:1 weight ratio in a plastic container. 
SECTION: HDMI Sequencing
10.2. Using a spatula, the components were mixed thoroughly till the PDMS become milky white texture.
SECTION: HDMI Sequencing
10.3. To remove the air bubble from the PDMS mix, it is degassed in a vaccum chamber for a total duration of 120 min.
SECTION: HDMI Sequencing
10.4. Depending of the length of the biopsy needle, pour the degassed PDMS to a petridish to the appropriate level and incubate in a 80 °C oven for a total duration of 120 min to polymerize PDMS.
SECTION: HDMI Sequencing
10.5. Using a fine spatula (Sigma Aldrich, Cat. No. Z193216-2EA), pry out the polymerized PDMS block out of the petridish and place on aluminum foil.
SECTION: HDMI Sequencing
10.6. Using a disposable biopsy punch (World Precision Instrument; Cat. No. 504647), create 1.5-2 mm PDMS punches. With help of fine tweezers, use these punches to block the inlet and the outlet of the NovaSeq HDMI flow cell.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
11. Before Starting
SECTION: Flow cell cleanup &amp; Enzymatic treatment
11.1. Thaw the following components:
10x rCutSmart buffer
Switch on the incubator to 37 °C.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
11.2. Prepare the 1X rCutSmart buffer. Mix the content well.

 

SECTION: Flow cell cleanup &amp; Enzymatic treatment
11.3. Prepare the DraI mix (without enzyme). Mix the content well.

 

SECTION: Flow cell cleanup &amp; Enzymatic treatment
11.4. Incubate both of the above buffer (without the enzyme, marked with **) at 37 °C for at least 30 min
SECTION: Flow cell cleanup &amp; Enzymatic treatment
12. Remove the sequencing reagents from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator. Wash the flow channels of the sequencing flow cell with 200 µL of NFW. Repeat this step for a total of 3 times.

 

Between each wash, the liquid in the channels are completely aspirated using vacuum liquid aspirator. The channels should be dry after the aspiration. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
13. Remove the NFW from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator. Wash the flow channels of the sequencing flow cell with 140 µL of 1X rCutSmart buffer.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
14. Add 60 µLof DraI Enzyme to the DraI mix (enzyme marked in **) above to the buffer mix. Mix the content well using a P1000 pipette. Remove the fluid from from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator and load the flow cell channels with140 µL DraI mix cocktail. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
15. Absorb the excess fluids from the inlet & outlet of the sequencing flow cell and using a precision forcep (VWR Cat. No. BOCH1930), block the inlets and & outlets using the 1.5-2mm PDMS biopsy cores to prevent evaporation of the reagents. Apply scotch tape to ensure no evaporation losses.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
16. Place the flow cell into a humidification chamber made with a large square petridish with some tissue towel paper soaked in NFW. Seal the petridish with parafilm tapes to completely seal the large petridish. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
17. Incubate the flowcell in the sealed humidification chamber at 37 °C in an oven for a duration of 960 min to 1200 min . 

SECTION: Flow cell cleanup &amp; Enzymatic treatment
18. After 960 min of incubation, check the flow channel and if air bubble are found in the flow channels, repeat  with fresh  with DraI mix cocktail and incubate for an additional 240 min .
SECTION: Flow cell cleanup &amp; Enzymatic treatment
19. Before Starting
SECTION: Flow cell cleanup &amp; Enzymatic treatment
19.1. Thaw the following components:
10x Exonuclease I buffer
Switch on the incubator to 37 °C.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
19.2. Prepare the 1X Exonuclease I buffer. Mix the content well.

 

SECTION: Flow cell cleanup &amp; Enzymatic treatment
19.3. Prepare the Exonuclease I mix (without enzyme). Mix the content well.

 

SECTION: Flow cell cleanup &amp; Enzymatic treatment
19.4. Incubate both of the above buffer at 37 °C for at least 30 min. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
20. Remove the DraI mix cocktail from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator. Wash the flow channels of the sequencing flow cell with 200 µL of NFW. Repeat this step for a total of 3 times.

Between each wash, the liquid in the channels are completely aspirated using vacuum liquid aspirator. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
21. Remove the NFW from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator. Wash the flow channels of the sequencing flow cell with 130 µL of 1X Exonuclease I buffer.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
22. Add 30 µLof  to the Exonuclease I enzyme + 16.75 µL Quick CIP enzyme to the Exonuclease I mix above. Mix the content well using a P200 pipette. Remove the fluid from from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator and load the flow cell channels with140 µL Exonuclease I mix cocktail each. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
23. Absorb the excess fluids from the inlet & outlet of the sequencing flow cell and using a precision forcep, block the inlets and & outlets using the 1.5-2mm PDMS biopsy cores to prevent evaporation of the reagents. Apply scotch tape to ensure no evaporation losses.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
24. Place the flow cell into a humidification chamber made with a large square petridish with some tissue towel paper soaked in NFW. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
25. Incubate the flow cell in the humidification chamber at 37 °C in an oven for a duration of 45 min
SECTION: Flow cell cleanup &amp; Enzymatic treatment
26. After the incubation, check the flow channel and if air bubble are found in the flow channels, repeat  with fresh Exonuclease I mix cocktail and incubate for an additional 15 min.
SECTION: Flow cell cleanup &amp; Enzymatic treatment
27. Remove the Exonuclease I mix cocktail from the flow channels of the NovaSeq flow cell using vacuum liquid aspirator. Wash the flow channels of the sequencing flow cell with 200 µL of NFW. Repeat this step for a total of 3 times.

Between each wash, the liquid in the channels are completely aspirated using vacuum liquid aspirator. 
SECTION: Flow cell cleanup &amp; Enzymatic treatment
28. The enzymatically treated S4 flow cell can then be processed directly for the Nova-ST chip preparation, or it can also be stored safely at 4 °C  for at least 2 weeks (based on our experience). 

If the flow cell is going to be stored, replace the NFW from the flow channels with 200 µL of IDT TEpH 8 buffer. Absorb the excess fluids from the inlet & outlet of the sequencing flow cell and using a precision forceps, block the inlets and & outlets using the 1.5-2mm PDMS biopsy cores to prevent evaporation of the reagents. Apply scotch tape to ensure no evaporation losses and proceed with the storage.

Please read the "Guidelines & Warnings" section, before the glass cutting step to prepare Nova-ST chips.

SECTION: Manual cutting strategy for Nova-ST chip preparation
31. The orientation of the chip is marked with the QR code present in the corner of the NovaSeq flow cell. 


 

SECTION: Manual cutting strategy for Nova-ST chip preparation
32. The flow cell is removed from its plastic adapter by releasing the plastic clip holding the chip. The flow cell is then incubated at 50 °C for 20 min  to dry the flow channels.

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
33. Using a scalpel, gently pry between the thin & thick glass layers which is separated by a black gasket. This has to be done on all sides of the flow cell. By gentle prying, the layers would slowly separate on all the sides. Once the layers have separated significantly, hold them and gradually separate the layers by hand till the thin and thick layers have separated completely. 
 

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
34. After separating the layers, the glass slides are glued to paper masking tapes. The excess paper tapes around the glass slides are trimmed away.

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
35. The scoring pattern was created using the machine suppliers dedicated software, Carbide Create. The glass plates dimensions were defined in the software as well as the desired scoring pattern. The pattern used for scoring both the glass slides:

 


 

SECTION: Manual cutting strategy for Nova-ST chip preparation
36. The glass slides were aligned and fixed onto the bread-board stage of the instrument. As per manufacturers protocol, the x, y and z coordinates of the spindle is set. The scoring depths was then adapted according to the glass thickness and the direction. The thicker glass plate (1.2mm thick) was scored with a 0.6mm depth in the width direction (shortest side) and 0.2mm depth in the length direction. For the thin section (thickness 0.3mm), depths of 0.4mm and 0.1mm were used, respectively. Each score was performed with a single pass of the tool.

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
37. Retrieve the glass slides from the CNC machine. Using the glass running pliers (SPEEDEOX), place the glass slides such that the score lines align with the reference line on the pliers. Adjust the set screws of the pliers to control the width of the jaws such that the glass layer sits flushed against the jaws when held between it. Apply gentle pressure, to break the glass along the score lines. Firstly, break the glass slide along the width (shorter length). Then, break along the long score lines to produce 1cm2 Nova-ST chip tiles.

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
38. Prepare 4x 24-well plate for transferring the chips. After cutting the chips, with help of sharp forceps gently remove the Nova-ST chips from the adhesive tape, without damaging the functional surface of the Nova-ST chip. Transfer the Nova-ST chips to the wells of 24 well plate according to an example color scheme described below:
 
The wells where the Nova-ST chips are stored are labeled according to their location in the S4 flowcell. This is needed for the identification of the location of the chips in the original NovaSeq flow cell. Example, the Nova-ST chip 1A1 is derived from the thick glass slide and is closest to the outlet port.

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
39. After the chip transfer to the corresponding 24 well plates are completed, proceed with the wash steps below:
SECTION: Manual cutting strategy for Nova-ST chip preparation
39.1. Using a multi-channel pipette, add 1 ml of NFW to the wells of the 24 well plate. After wash, discard NFW to a collection reservoir.

 
repeat the step for a total of 3X times.
SECTION: Manual cutting strategy for Nova-ST chip preparation
29. Before Starting:
SECTION: Manual cutting strategy for Nova-ST chip preparation
30. If the NovaSeq flow cell after the enzymatic treatment has been stored at 4 °C , remove the PDMS plugs and wash the flow channels of the sequencing flow cell with 200 µL of NFW. Repeat this step for a total of 3 times.

Between each wash, the liquid in the channels are completely aspirated using vacuum liquid aspirator. 
SECTION: Manual cutting strategy for Nova-ST chip preparation
29.1. Clean all the surfaces and tools being used for preparing the Nova-ST with 70% Ethanol followed by RNA zap and DNA zap.
SECTION: Manual cutting strategy for Nova-ST chip preparation
29.2. Prepare 0.1N NaOH solution

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
29.3. Prepare 0.1M Tris 7.5 solution

 

SECTION: Manual cutting strategy for Nova-ST chip preparation
39.2. Using a multi-channel pipette, add 1 ml of 0.1N NaOH solution to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. Incubate the chips for 5 min at Room temperature . After incubation discard the 0.1N NaOH solution from the wells to a collection reservoir. After each wash remove as much as liquid possible from the wells.

repeat the step for a total of 3X times.

SECTION: Manual cutting strategy for Nova-ST chip preparation
39.3. Using a multi-channel pipette, add 0.1M Tris 7.5 solution to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. After incubation discard the 0.1M Tris 7.5 solution from the wells to a collection reservoir. After each wash remove as much as liquid possible from the wells.

repeat the step for a total of 3X times.
SECTION: Manual cutting strategy for Nova-ST chip preparation
39.4. Using a multi-channel pipette, add 1 ml of 1x IDT TE pH 8  to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. After wash, discard the TE buffer to a collection reservoir.
SECTION: Manual cutting strategy for Nova-ST chip preparation
39.5. Using a multi-channel pipette, add 1 ml of 1x IDT TE pH 8 to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. Seal the 24 well plate with parafilm M tapes and store the chips at 4 °C .
SECTION: Automatic dicing strategy for Nova-ST chip preparation
40. Before Starting:
SECTION: Automatic dicing strategy for Nova-ST chip preparation
40.1. Clean all the surfaces and tools being used for preparing the Nova-ST with 70% Ethanol followed by RNA zap and DNA zap.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
40.2. Prepare 0.1N NaOH solution

 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
40.3. Prepare 0.1M Tris 7.5 solution

 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
41. If the NovaSeq flow cell after the enzymatic treatment has been stored at 4 °C , remove the PDMS plugs and wash the flow channels of the sequencing flow cell with 200 µL of NFW. Repeat this step for a total of 3 times.

Between each wash, the liquid in the channels are completely aspirated using vacuum liquid aspirator. 
SECTION: Automatic dicing strategy for Nova-ST chip preparation
42. The orientation of the chip is marked with the QR code present in the corner of the NovaSeq flow cell.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
43. The NovaSeq flow is mounted on dicing tape which has a sticky backing that holds the flow cell on a thin sheet metal frame, to prepare for dicing process. The thick glass side of the flow cell is glued on to the adhesive film. In order to reduce the adjustment for alignment in the dicing machine, the flow cell is glued on to the dicing tape, pre-aligned. 
SECTION: Automatic dicing strategy for Nova-ST chip preparation
44. The metal frame with flow cell is fixed to the dicing stage. The x-,y- and   alignment is performed. The origin for the start of the dicing is initiated. Cutting speed is set to 1 mm/s and dicing process is initiated. 

 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
49. Once the layers have been separated, place the separated chip on a paper towel with the functional surface facing up. After separating a batch of chips, using a sharp forceps, transfer the chips to the respective 24 well plate, with the functional surface of the chips facing upwards.

 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
50. After all the chip transfer has been performed to the corresponding 24 well plates, proceed with the wash steps below:
SECTION: Automatic dicing strategy for Nova-ST chip preparation
50.1. Using a multi-channel pipette, add 1 ml of NFW to the wells of the 24 well plate. After wash, discard NFW to a collection reservoir.

 
repeat the step for a total of 3X times.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
50.2. Using a multi-channel pipette, add 0.1N NaOH solution to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. Incubate the chips for 5 min at Room temperature. After incubation discard the 0.1N NaOH solution from the wells to a collection reservoir. After each wash remove as much as liquid possible from the wells.

repeat the step for a total of 3X times.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
50.3. Using a multi-channel pipette, add 0.1M Tris 7.5 solution to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. After incubation discard the 0.1M Tris 7.5 solution from the wells to a collection reservoir. After each wash remove as much as liquid possible from the wells.

repeat the step for a total of 3X times.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
50.4. Using a multi-channel pipette, add 1 ml of 1x IDT TE pH 8 to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. After the wash, discard the TE buffer to a collection reservoir.
SECTION: Automatic dicing strategy for Nova-ST chip preparation
50.5. Using a multi-channel pipette, add 1 ml of 1x IDT TE pH 8 to the wells of the 24 well plate. Ensure all the chips are completely submerged in the liquid. Seal the 24 well plate with parafilm M tapes and store the chips at 4 °C .
SECTION: Automatic dicing strategy for Nova-ST chip preparation
45. Firstly, the dicing is performed along the length of the NovaSeq flow cell and its cut into slabs of 1 cm thick. Without detaching the separated 1 cm slab from the dicing tape, the tape assembly is turned by 900 angle and the dicing is repeated along the width of the NovaSeq flow cell and it cut into desired dimensions. An example of a recent dicing pattern used by our lab:

 
 
 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
46. After the dicing steps are completed, the diced NovaSeq flow cell is removed from the instrument and the dicing tape is cut out to retrieve the diced NovaSeq flow cell. 
SECTION: Automatic dicing strategy for Nova-ST chip preparation
48. With help of sharp forceps gently remove the Nova-ST chips from the adhesive dicing tape. The Nova-ST chips at this stage is still bonded with the thin and thick sections still bonded. Using a fresh razor, pry gently at the side between the thin and thick section (as shown below). A gentle push is sufficient to separate the layers. Ensure not to disturb/damage the function surface of the Nova-ST chips.


 

SECTION: Automatic dicing strategy for Nova-ST chip preparation
47. Prepare 6x24-well plate for transferring the chips. For the dicing schema described in step 46, the following plate configuration is used:

 
The wells where the Nova-ST chips are stored are labeled according to their location in the S4 flowcell. This is needed for the identification of the location of the chips in the original NovaSeq flow cell. Example, the Nova-ST chip 3A1 is derived from the thick glass slide and is closest to the outlet port.
SECTION: Data processing
51. Details on data processing, post HDMI sequencing can be found here https://github.com/aertslab/Nova-ST