Preliminary

• book the Cluster Station (CS) beforehand;
• estimate the optimal concentration of DNA library (default concentration - 4pM; if the library was already sequenced, determine the concentration according to the number of clusters in the previous runs; optimal number of clusters - 7,5mln per lane);
• check, that you have:
  • 5M Betaine, 12ml (should be prepared at least once a month, 50ml at a time);
  • empty flowcell for washing manifolds in use;
• wash the CS (steps 1-4 of Cluster preparation);
• on the way back from CS take the "cluster preparation kit" box from -80°C;

Buffers

1. [  ]  prepare and label:
   • 8-strips:
     • #A — hybridization buffer,
     • #B — template (write the number of library on each tube),
     • #C — wash buffer,
     • #D — amplif. premix,
     • #E — extension mix,
     • #0 — empty;
   • 1,7ml eppendorf tube:
     • #IEM — initial extension mix,
   • 50ml tubes:
     • #1 — Bst mix,
     • #9 — formamide,
     • #10 — wash buffer,
     • #11 — ampl. premix,
     • #12 — storage buffer,
     • #X — ampl. premix preparation;


2. [  ]  fill tubes and store them on ice:
   • #9 — 15ml formamide,
   • #10 — 10ml wash buffer,
   • #12 — 10ml storage buffer,
   • #A — 8 x 140µl hybridization buffer,
   • #C — 8 x 100µl wash buffer,


3. [  ]  prepare amplification premix:

   H2O	15ml
   Cluster buffer	3ml
   5M Betaine	12ml
   total	30ml

   • mix, vortex,
   • 5min 0°C (the solution should become clear),
   • filter through 0,2µm filter,
   • transfer:
        12ml in #11(ampl. premix),
        12ml in #1(Bst. mix),
        8 x 100µl in #D(amplif. premix);


4. [  ]  prepare IEM (Initial Extension mix):

   Amplification premix	975µl
   10mM dNTP's	20µl
   Taq polymerase	5µl
   total	1ml

   • transfer 8 x 120µl in #E(extension mix);


5. [  ]  prepare Bst-amplification mix (tube #1):

   Amplification premix	12ml
   10mM dNTP's	240µl
   Bst polymerase	120µl
   total	12,36ml



6. [  ]  prepare template (should be 120µl of template per each channel)
   

   EB Qiagen	17µl
   10nM library	1µ2
   2N NaOH	1µl
   total	20µl

   • vortex, spin down, 5min RT,
   • prepare tubes with #HB — hybridization buffer (one per library),
   • transfer ?µl of denaturated library to ?µl of hybridization buffer #HB (for example: (i) 4µl to 996µl to prepare 4pM solution for 8 channels; (ii) 2µl to 398µl to prepare 5pM solution for 1 channel),
   • vortex, spin down, transfer 120µl of each library in #B(template),
   • keep strip #B(template) on RT till loading on the flowcell.

Equipment and consumables

• powder-free gloves;
• 1ml automatic pipette with tips (for emergency cases);
• 200µl automatic pipette with tips (for emergency cases);
• lens cleaning tissue;
• ~250ml of clean H₂O;
• washing bottle with clean H₂O;
• 100ml glass beaker for rinsing ends of tubes;
• hybridization manifold;
• amplification manifold;
• empty flowcell;
• new flowcell;
• plastic tweezers;
• 2x 50ml tube holders;
• 2ml Eppendorf tube holder;
• marker pen;
• pen
• this protocol + cluster preparation checklist

Cluster amplification

1. turn on CS, wait 30 s, turn on computer;


2. empty the waste container, wipe the table and the CS with a wet tissue;


3. depending on the situation:
      if CS was previously used by us:
   • change water in old water storage tubes (WST),
   • start wash protocol;
   
      if CS was previously used by another group:
   • change WSTs to new ones with fresh water (with tubings washing and air priming),
   • start wash protocol;
   
      if CS was previously washed with decon by another group:
   • for 50ml and 15ml tubes: shake the old WSTs gently to wash also the cap, change water in old WSTs and shake again, remove drops of water from the caps by 1ml pipette, change WSTs to new ones with fresh water (with tubings washing and air priming),
   • for 2ml tubes: change wash caps twice by pouring/gathering water with 1ml pipette,
   • start wash protocol;


4. depending on the plans:
      if only cluster amplification will be performed:
   • check, that tubes 1,9,10,11,12 are filled with H₂O;
   • open "Amplification_only" recipe, "WashAmponlyLines", "Start", "OK", ~10min;
   
      if linearisation/blocking/primer hybridisation will be performed:
   • check, that tubes 5,7,10,12,14,15,17,18 are filled with H₂O;
   • open "###" recipe, "###", "Start", "OK", ~10min;
   
      if full protocol will be performed:
   • check, that tubes 1,3,5,7,9,10,11,12,14,15,17,18 are filled with H₂O;
   • open "###" recipe, "###", "Start", "OK", ~15min;


5. "User Wait" on the top & follow messages on the screen:
   • remove H₂O from tubes in reagent positions 1,9,10,11,12 (remove drops from the cap by a pipette)
     prime air 1,9,10,11,12;
   • load reagents in positions 1,9,10,11,12
   • air gap [26 · 40µl/min · 130µl];
   • remove washing bridge;
   • load flowcell, tube strip holder, hybridization manifold;
   • load strip #A(hybridization buffer), Ok;
     CHECK!!! that all channels behave similarly,
     [60µl/min · 120µl];
   • strip #B(template), Ok (time for heating),
     96°C · 1°C/s
     [15µl/min · 75µl] 5 min;
     [100µl/min · 10µl];
     wait 30s
     40°C · 0.05°C/s (time for cooling)
   • strip #C(wash buffer), Ok,
     [15µl/min · 75µl] 5 min;
     40°C · 1°C/s
   • strip #D(amplif. premix), Ok,
     [15µl/min · 70µl] 5 min;
   • strip #E(extension mix), Ok,
     [60µl/min · 95µl] 1,5 min;
     74°C · 1°C/s
     wait 90s
     60°C · 1°C/s
   • strip #0(empty tube), Ok,
     [15µl/min · 20µl];
   • remove hybridization manifold (put it on clean surface), connect amplification manifold;
   • Ok & check for proper flow through the flowcell and manifold;
     prime reagents 1,9,10,11,12;
     

     35 cycles:

     #9(formamide) [30µl/min · 28µl]
     #11(ampl. premix) [30µl/min · 28µl]
     #1(ampl. mix) [30µl/min · 36µl]

     
     #10(wash buffer) [30µl/min · 140µl],
     20°C · 1°C/s,
     #12(storage buffer) [15µl/min · 95µl],
     20°C · 1°C/s;
   
   
6. There are several possibilities to proceed:
   • cluster staining (go to §4)
   • flowcell storage
   • primer hybridization (go to §3)

Flowcell storage

1. air gap [26 · 30µl/min · 40µl];
2. unclip "reagent delivery port" & "central clamp" of amplification manifold;
3. accurately clean flowcell with water and lens cleaning tissue;
4. put flowcell in 50ml tube with Storage buffer, store at 4°C;


wash hybridization and amplificvation manifolds:
5. put a special empty FC and attach the manifold;
6. clean manifold with water [3 · 60µl/min · 240µl];
7. air [26 · 40µl/min · 200µl];
8. remove manifold & empty FC;
9. go to "CS cleaning after amplification".

CS cleaning after amplification

1. attach washing bridge;
2. pour fresh water in water-tubes 1,9,10,11,12;
3. for all tubes in positions X = 1,9,10,11,12:
   • remove tube with reagent,
   • air gap [X · 240µl/min · 30µl],
   • rinse the end of the tubing using washing bottle,
   • attach tube with water;
4. recipe window: "WashAmponlyLines", "Start", "OK" (about 10min);
5. close the program; turn off computer, turn off CS

Notes

5M Betaine

•    recipe for 250ml 5M Betaine:
     anhydrous Mr=117,15: 146,4g Betaine + 121,85ml H₂O
     monohydrate Mr=135,2: 169,1g Betaine + 99,35ml H₂O
  dissolve,
  heat at 37°C 1h,
  filter through 0,2µm filter,
  store at 4°C;



• volumes of reagents after reaction:
  • #A: ~160µl in 8 tubes;
  • #C: ~150µl in 8 tubes;
  • #D: ~190µl in 8 tubes;
  • #E: ~130µl in 8 tubes;
  • #1: ~4,5ml;
  • #9: ~8ml;
  • #10: ~7,5ml;
  • #11: ~5ml;
  • #12: ~4,5ml;
  • A: ~160µl in 8 tubes;