TITLE: Single-strand library preparation protocol
AUTHORS: Tomasz Suchan

[DESCRIPTION]
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[STEPS]
?. [ Guanidine tailing]
Prepare master mix 2 (assemble at room temperature, can precipitate when on ice):
[water]
[NEBuffer 4 (10x)]
[CoCl2 (2.5 mM)]
[GTP (100 mM)]
[TdT (20 U/μl)]
?. [Second strand synthesis]
Prepare master mix 3:
[water]
[NEBuffer 4 (10x)]
[dNTP mix (25 mM each)]
[P2-CCCCC oligo (15 mM)]
[Klenow exo- (5 U/μl)]
?. [Blunt-end reaction]
Prepare master mix 4:
[water]
[NEBuffer 4 (10x)]
[BSA (10 mg/ml)]
[T4 DNA polymerase (3 U/μl)]
?. [Ligation of P1 adapter]
Prepare barcoded P1 adapters (25 μM concentration).
Each sample should be mixed with a different adapter in case the barcoded adapters are used.
?. [Dephosphorylation]
Prepare master mix 1:
[NEBuffer 4 (10x)]
[water]
[FAST alkaline phosphatase (1 U/μl)]
?. [Dephosphorylation]
Add 3 μl of master mix 1 to 10 μl of DNA (total volume = 13 μl).
?. [ Guanidine tailing]
Add 7 μl of the master mix 2 to the denatured DNA (total volume = 20 μl).
?. [ Guanidine tailing]
Incubate for 30 min at 37°C, heat-kill the enzymes for 10 min at 70°C. Spin down.
?. [Second strand synthesis]
Add 10 μl of the master mix 3 to 20 μl of the DNA (total volume = 30 μl).
?. [Second strand synthesis]
Incubate at room temperature for 3 hours, heat-kill enzymes for 20 min at 75°C. Spin down.
?. [Blunt-end reaction]
Add 5 μl of the master mix 4 to 30 μl of the DNA (total volume = 35 μl).
Keep both DNA and the mix on the ​ice when assembling the reaction.
?. [Blunt-end reaction]
Incubate for 15 min at 12°C. Hold at 4°C and proceed directly to the next step.
?. [Blunt-end reaction]
Perform AMPure cleanup with the beads:sample ratio 2:1 (70 μl of the beads and 35 μl of the reaction) according to the manufacturer’s instructions. Resuspend in 11 μl of 10 mM Tris or water.
?. [Ligation of P1 adapter]
Prepare master mix 5:
[water]
[PEG-4000 (50%)]
[T4 ligase buffer (10x)]
[T4 DNA ligase (400 U/μl)]
?. [Ligation of P1 adapter]
Add 1 μl of the adapter (25 μM working solution) to 10 μl of the DNA. Briefly vortex and spin.
?. [Ligation of P1 adapter]
Add 9 μl of the master mix 5 to each sample (total volume = 20 μl).
?. [Ligation of P1 adapter]
Incubate at 16°C for 3h. Spin down.
?. [Ligation of P1 adapter]
Perform AMPure cleanup with the beads:sample ratio 1:1 (20 μl of the beads and 20 μl of the reaction) according to the manufacturer’s instructions. Resuspend in 11 μl of 10 mM Tris or water.
?. [Ligation of P1 adapter]
Transfer 10 μl the purified DNA to the plate/tubes.
?. [PCR amplification]
Prepare master mix 4:
[water]
[Q5 polymerase buffer (5x)]
[dNTP mix (25 mM each)]
[Q5 hot-start polymerase]
?. [PCR amplification]
Add 35 μl of the master mix 3 to each tube/well.Add 2.5μl of each primer (5 μM working solutions).Add 10 μl of the template.
?. [PCR amplification]
Run PCR program:30 s at 98°C15 cycles of:10 s at 98°C20 s at 60°C25 s at 72°C5 min at 72°Chold at 4°C
Determine experimentally the required number of PCR cycles.
?. [PCR amplification]
Check reaction on a gel or Tapestation/Fragment Analyzer. Quantify using Qubit fluorimeter.
?. [Dephosphorylation]
Incubate for 1 h at 37°C, denature the DNA for 5 min at 95°C, put immediatly on ice.
Prepare ice at the and of incubation. Move sample immediatelly form 95°C to the ice.