Preliminary

• check the disk space (should be ### on drive D);
• prepare PE Cluster Generation Kit Box 2 (-20°C) and Box 4 (NT);
• thaw all buffers from the Box 2 and 12ml aliquot of 5M Betaine;
• write (i) date and (ii) FlowCell number on both boxes (2 & 4);

Prepare reagents

1. label 13x 15ml tubes:

   #9	deprotection premix
   #10	deprotection mix
   #11	linearization 2 mix
   #12	blocking mix
   #13	Bst mix
   #14	cluster premix
   #15	formamide
   #16	read 2 sequencing primer mix
   #17	linearization 2 Buffer
   #18	1x blocking buffer
   #19	0.1N NaOH
   #20	TE buffer
   #21	wash buffer



2. sign the tube with 5M Betaine "Cluster premix";



3. prepare kits:
   • buffers: vortex briefly and spin down;
   • enzymes: spin down, mix by finger tipping, spin down again & put in -20°C box;


4. fill tubes:

   #21	wash buffer, 10ml;
   #19	NaOH, 0.1N, 4ml;
   #20	TE buffer, 3ml (the whole amount);
   #15	formamide, 8ml;



5. prepare:
   • deprotection premix in tube #9:

     2ml 5x deprotection buffer 400µl H2O 1.6ml / 2x800µl

     
     
   • deprotection mix in tube #10:

     2ml 5x deprotection buffer 400µl H2O 1.56ml / 2x780µl --- mix --- deprotection enzyme 40µl --- mix ---

     
     
   • cluster premix:

     conc. stock 30ml Betaine 2M 5M 12ml / 12.88g H2O   mQ 15ml cluster buffer 1x 10x 3ml

     • vortex;
     • 5min at 0°C (the solution should become clear);
     • filter through 0.2µm Steriflip unit;
     • transfer 10ml in tube #14;
     
     
   • Bst mix in tube #13:

     conc. stock 10.3ml cluster premix 1x 1x 10ml dNTP 194µM 10mM 200µl Bst DNA polymerase 78u/ml 8u/µl 100µl

     
     
   • linearization 2 buffer in tube #17:

     conc. stock 2ml linearization 2 buffer 1x 10x 200µl H2O   mQ 1.8ml / 2x900µl

     
     
   • linearization 2 mix in tube #11:

     conc. stock 2ml H2O   mQ 1.68ml / 2x840µl linearization 2 buffer 1x 10x 200µl BSA     20µl --- mix --- linearization 2 enzyme     100µl --- mix ---

     
     
   • 1x blocking buffer in tube #18:

     conc. stock 5ml blocking buffer 1x 10x 500µl H2O   mQ 4.5ml / 5x900µl

     
     
   • blocking mix in tube #12:

     conc. stock 2.02ml blocking buffer (from #18) 1x 1x 1.82ml / 2x910µl ddNTP 99µM 2.5mM 80µl Blocking enzyme A     24µl Blocking enzyme B     100µl

     
     
   • read 2 sequencing primer mix in tube #16:

     1.5ml hybridization buffer 1.49ml / 2x747µl read 2 PE seq primer 7.5µl

   
   

Cluster regeneration

1. load reagents on the PE module (keep water tubes in rack);
2. place the waste tube into the waste container;
3. click OK to resume the "GA2-PEM_2x36_PE_v4" recipe and start read 2;
NB! cluster regeneration takes ~5 hours, meanwhile prepare the sequencing reagents (see "Preparation of sequencing reagents" protocol)
4. check volumes of reagents after reaction:

   position reagent volume initial initial should remain #9 deprotection premix 2ml 1.4ml 800µl #10 deprotection mix 2ml 1.4ml 800µl #11 linearization 2 mix 2ml 1.4ml 800µl #12 blocking mix 2.02ml 1.42ml 300µl #13 Bst mix 10.3ml 9.7ml 5.38ml #14 cluster premix 10ml 9.25ml 4.69ml #15 formamide 8ml 7.25ml 3.89ml #16 read 2 sequencing primer mix 1.5ml 750µl 150µl #17 linearization 2 buffer 2ml 1.25ml 650µl #18 blocking buffer, 1x 3.18ml 2.43ml 1.83ml #19 NaOH, 0.1N 4ml 3.25ml 2ml #20 TE buffer 3ml 2.25ml 1ml #21 wash buffer 10ml 9.05ml 3.49ml

Sequencing 2

1. exchange the sequencing reagent tubes used for read 1 with fresh ones in order: 1,3,4,5,7,6;
2. click OK to resume the "GA2-PEM_2x36_PE_v4" recipe;
        first base incorporation begins
3. remove reagent tubes from PE module, positions 9-21:
   • remove reagent tube;
   • wash tubing from washing bottle;
   • reinstall 15ml tube with ~10ml of mQ from the pre-run wash step;
4. when 1^st base incorporation is complete, click Cancel to perform refocusing:
   • open "Manual Control/Setup" window;
   • pump: [to: flowcell · solution: 3 · volume: 100 · asp. rate: 250 · disp. rate: 2500] ⇒ Enter;
   • perform refocusing (only Z, do not change XY settings, "Focusing", p.8-9: setting Z roughly & accurately) and recalibrate the autofocus laser;
5. click OK to resume the "GA2-PEM_2x36_PE_v4" recipe;
        imaging begins
6. evaluate the first base report data:
   * number of clusters should be 90-120x10³ per tile;
   * FC tilt: difference between min and max focus stage values should be less than 15000 nm;
   * intensity values:

   Confidence A & C G T High > 650 > 1000 > 1200 Reasonable > 350 > 650 > 700 Low < 250 < 350 < 400

7. click OK to process sequencing;

After run

1. perform the post-run wash ("GAII maintenance" protocol);
2. weigh reagent bottles and record results.