Surface amplification

Introduction

This protocol describes the materials and methods to generate surface amplified sequencing library on a reservoir.

Circular single stranded library hybridizes to surface immobilized P7 oligonucleotides on sequencing reservoir. These oligonucleotides serve as forward primers to initiate rolling circle amplification (RCA) to generate sequencing clusters. The forward strand will contain P5’ sequences, which will anneal to the surface immobilized P5 oligos and allow rolling circle amplification to produce the reverse strand. Hyperbranched clusters are produced by forward and reverse RCA products annealing to the surface and extending.

Once hyperbranched clusters are generated by RCA, Thermolabile USER II is used to cleave the reverse strands from the surface. After denaturing and washing, the sequencing reservoir surface contains thousands of forward strand RCA clusters.

Since the TIRF system is so sensitive to incorporation at the sequencing reservoir surface, TdT is used to terminate any 3’ ends. After a last set of denature and wash steps, the sequencing reservoir is ready to sequence.

Revision history

Materials and equipment

Notes before starting

Optimal circular library hybridization concentration is critical to achieving a high density of sequencing clusters without compromising monoclonality. We have achieved good results hybridizing clusters at concentrations of 0.15-0.4 pM, but ideal concentrations should be determined empirically.

Sodium hydroxide dilutions should be made fresh for same day use.

Procedure

1. Prepare reservoir for hybridization

1. Dispense 100 µL nuclease free (NF) H₂O into a new reservoir, being careful not to contact glass surface at bottom of reservoir
2. Aspirate and discard the liquid from the reservoir with P-100 or P-200 pipette by tilting the reservoir opening towards you and carefully placing the tip of the pipette at the lowest edge of the reservoir

2. Hybridize library

1. Dilute library to loading concentration
   • Library should be at 0.15 - 0.4 pM in 2x SSC + 0.1% Triton X 100 (SSCT)
   • Ensure there is at least 100 µL of diluted library per reservoir
2. Dispense 100 µL diluted library into reservoir
3. Incubate reservoir at 65º C for 4 minutes, 40º C for 44 minutes, and then room temperature for 4 minutes
4. After final 4 minute room temperature incubation, remove and discard 100 µL solution from reservoir
5. Dispense 50 µL of 1x RCA Buffer into reservoir

3. Cluster generation

1. Make Phi29XT Reaction Mix using the table below, adding each component:

   • Reagent	Volume for 1 reservoir	Final concentration
     Nuclease free water	60 µL
     Phi29-XT Reaction Buffer, 5x	20 µL	1x
     dNTP Solution Mix, 10 mM	10 µL	1 mM
     Phi29XT Polymerase, 10x	10 µL	1x
     Total volume	100 µL

   • Vortex and spin down reaction mix, keep on ice
2. Remove 1x RCA buffer from sequencing reservoir by tilting reservoir towards you and aspirating at the lowest edge of the reservoir with a p-100 or p-200 pipette
3. Dispense 50 µL of the Phi29XT Reaction Mix into the reservoir
   1. Ensure liquid is at bottom of reservoir
   2. Store remaining reaction mix at 4º C for a reaction refresh
4. Incubate reservoir at 42º C for 30 minutes
5. Refresh Phi29XT Reaction Mix:
   1. After 30 minutes, remove 20 µL of Phi29XT Reaction Mix from reservoir
   2. Dispense the 50 µL of Phi29XT Reaction Mix remaining from step 3.3.2 into the reservoir and gently swirl
   3. Ensure all liquid is at bottom of reservoir and incubate at 42º C for an additional 2 hours
   4. After 2 hour incubation, proceed directly to step 4

4. Cleave reverse strands

1. Begin incubating the reservoir with Phi29XT Reaction Mix at 37º C
2. Dispense 2 µL of Thermolabile USER II enzyme into the reaction mix, pipetteing up and down to ensure full volume is dispensed
3. Tilt the reservoir towards you and use a P-100 or P-200 pipette set to 50 µL to gently and carefully mix the liquid without contacting the bottom surface of reservoir
4. Incubate the reservoir at 37º C for 30 minutes
5. 4.5. After 30 minutes, remove the reaction mix
6. Denature reverse strands
   1. Dispense 100 µL of 100 mM NaOH into reservoir
      • Incubate 3 minutes at room temperature
   2. Remove NaOH from reservoir
   3. Rinse reservoir:
      1. Dispense 200 µL NF H₂O into reservoir
      2. Pipette up and down 3 times to agitate the surface of the reservoir with the liquid
      3. Remove NF H₂O
   4. Repeat step 4.6.3 two times for a total of three rinses
7. Dispense 100 µL of 2x SSC + 0.1% Triton X 100 into reservoir

5. Block surface

1. Albumin blocking:
   1. Prepare albumin blocking solution using the table below:

      • Reagent	Volume for 1 reservoir	Final concentration
        Nuclease free water	42.5 µL
        Terminal Transferase Buffer, 10x	5 µL	1x
        Recombinant Albumin, 20 mg/mL	2.5 µL	1.12 mg/mL
        Total volume	50 µL

   2. Vortex and spin down mixture
   3. Remove liquid from reservoir
   4. Dispense 50 µL of albumin bocking solution into reservoir
   5. Incubate 10 minutes at room temperature
2. 3’ end blocking:
   1. Prepare terminal transferase (TdT) Reaction Mixture using table below:

      • Reagent	Volume for 1 reservoir	Final concentration
        Nuclease free water	35 µL
        Terminal Transferase Buffer, 10x	5 µL	1x
        CoCl₂, 10x	5 µL	1x
        Dideoxynucleotide Solution, 2.5 mM (0.625 mM each ddATP, ddCTP, ddGTP, ddTTP)	4 µL	0.2 mM
        Terminal Transferase, 20,000 U/mL	1 µL	400 U/mL
        Total volume	50 µL

   2. Vortex and spin down mixture
   3. Remove albumin blocking solution from reservoir
   4. Dispense 50 µL TdT Reaction Mixture into reservoir
   5. Ensure all liquid is at bottom of reservoir
   6. Incubate at 37º C for 30 minutes
3. Denature protein and rinse reservoir
   1. Remove reaction mix from reservoir
   2. Dispense 100 µL of 100 mM NaOH into reservoir
      • Incubate 3 minutes at room temperature
   3. Remove NaOH from reservoir
   4. Rinse reservoir:
      1. Dispense 200 µL NF H₂O into reservoir
      2. Pipette up and down 3 times to agitate the surface of the reservoir with the liquid
      3. Remove NF H₂O
   5. Repeat step 5.3.4 two times for a total of three rinses
4. Dispense 100 µL of 2x SSC + 0.1% Triton X 100 into reservoir