TITLE: Single_Stranded_Library_Workflow_Gut_Virome
AUTHORS: Xichuan Zhai

[DESCRIPTION]
Protocol of single-stranded library (SSLR) preparation for virome study

[BEFORE_START]
Fill bucket with crushed ice.

[GUIDELINES]
This protocol is designed for virome library preparation with our lab-customed single-stranded library (SSLR). This workflow includes 7 FLEXIBLE steps, virome isolation and extraction, mock community (positive control) preparation, reverse transcription with the purpose of dsRNA denatures, genome fragmentation, ligation with customed designed adaptors and library preparation for sequencing. Detailed information can be found on the next page.

[STEPS]
SECTION: Step1: Virome isolation and extraction
1. Checklist before starting 
 

SECTION: Step2: Heat treatment
2. Checklist before starting 
 

SECTION: Step3: Reverse transcription (RT)
3. Checklist before starting 
 

SECTION: Step4: Fragmentation
4. Checklist before starting 
 

SECTION: Step5: Ligation
5. Checklist before starting 
 

SECTION: Step6: Index PCR
6. Checklist before starting 
 

SECTION: Step7: AMPure beads clean up and library quality check
7. Checklist before starting
 

SECTION: Step1: Virome isolation and extraction
1.1. Make aliquots of  (procedure can be found from: dx.doi.org/10.17504/protocols.io.b2qaqdse).
SECTION: Step1: Virome isolation and extraction
1.2. Add 1 µLof 100-time diluted nuclease (check the stock for the dilution in SM buffer) to each sample and let them incubate at approximately 30 minat37 °Cper. sample. Vortex between each sample.
SECTION: Step1: Virome isolation and extraction
1.3. Consider increasing the incubation time if you expect a lot of external DNA.
SECTION: Step1: Virome isolation and extraction
1.4. Immediately after adding540 µL AVL buffer to inactivate nucleases.
SECTION: Step1: Virome isolation and extraction
1.5. Mix the mixture by pulse vortexing.
SECTION: Step1: Virome isolation and extraction
1.6. Incubate at Room temperature for 10 min.
SECTION: Step1: Virome isolation and extraction
1.7. Change gloves.
SECTION: Step1: Virome isolation and extraction
1.8. Briefly centrifuge the mixture with a microcentrifuge.
SECTION: Step1: Virome isolation and extraction
1.9. Add560 µLof absolute ethanol (96%) to the sample mixture.
SECTION: Step1: Virome isolation and extraction
1.10. CRITICAL STEP: Mix very well by pulse-vortex.
SECTION: Step1: Virome isolation and extraction
1.11. Briefly centrifuge the mixture with a microcentrifuge.
SECTION: Step1: Virome isolation and extraction
1.12. Add630 µLof the sample mixture to the spin column.
SECTION: Step1: Virome isolation and extraction
1.13. CRITICAL STEP: Do not touch the column rim with the pipet.
SECTION: Step1: Virome isolation and extraction
1.14. Centrifuge at 6,000 x g change the collection tube and repeat steps 1.12 to load all the extracts.
SECTION: Step1: Virome isolation and extraction
1.15. Add500 µLAW1 buffer to the spin column the following:

SECTION: Step1: Virome isolation and extraction
1.16. Centrifuge at6,000 x g, 1 min, change the collection tube.
SECTION: Step1: Virome isolation and extraction
1.17. Add 500 µLAW2 buffer to the spin column.
SECTION: Step1: Virome isolation and extraction
1.18. Centrifuge at20,000 x g, 3 min, change the collection tube.
SECTION: Step1: Virome isolation and extraction
1.19. Centrifuge at 20,000 x g, 1 min. Place the spin column in a low-binding RNAse-free 1.5 mL tube.
SECTION: Step1: Virome isolation and extraction
1.20. Add 30 µLof AVE (elution buffer) to the spin column and incubate Room temperaturefor1 min.
SECTION: Step1: Virome isolation and extraction
1.21. CRITICAL STEP: Pipet the AVE buffer directly onto the filter membrane without touching it with the pipet tip.
SECTION: Step1: Virome isolation and extraction
1.22. Centrifuge at6,000 x g, 1 minto collect the filtrates.
 

SECTION: Step2: Heat treatment
2.1. Preheated ThermoCycle machine to 95 °Cand sterilized PCR tubes or plates depending on the
how many samples are used for reverse transcription. 
SECTION: Step2: Heat treatment
2.2. Add16 µL in PCR tubes or platesand prepare on ice.
SECTION: Step2: Heat treatment
2.3. Briefly centrifuge the mixture with a microcentrifuge.
SECTION: Step2: Heat treatment
2.4. Put tubes or plates for heat treatment95 °Cfor3 min.
SECTION: Step2: Heat treatment
2.5. CRITICAL STEP: Transfer samples to ice immediately after3 min.

SECTION: Step2: Heat treatment
2.6. PAUSE POINT Ready for reverse transcription.
 

SECTION: Step3: Reverse transcription (RT)
3.2. Add 4 µLSuperScript‱ IV VILO‱ Master Mix to each sample and mix thoroughly, spin them down.
SECTION: Step3: Reverse transcription (RT)
3.3. Carefully place the PCR plate into the ThermoCycle machine and select the following program: 
 
 
SECTION: Step3: Reverse transcription (RT)
3.4. Purified with 1X AMPure XP beads (25 μL / 25 μL PCR reaction) and eluted with20 µLTris
buffer (10 mM, pH8.0) according to the purification procedures from .
 

SECTION: Step7: AMPure beads clean up and library quality check
7.1. Place AMPure XP beads into the Hula mixer to resuspend the beads and to equilibrate toRoom temperaturefor15 min. 
SECTION: Step7: AMPure beads clean up and library quality check
7.2. Label an Eppendorf tube or PCR plate with your sample ID and transfer the 25 µLPCR product.
SECTION: Step7: AMPure beads clean up and library quality check
7.3. Transfer20 µL(!!! 0.8X) of Beads solution to each PCR product (25 µL) and mix with 100 μL pipette tips (10 times up and down), resulting in a45 µLmixture.
SECTION: Step7: AMPure beads clean up and library quality check
7.4. Incubate for 5 minatRoom temperature.
SECTION: Step7: AMPure beads clean up and library quality check
7.5. Place the tube or plate with the mixture into the magnetic rack for 2-4 min, until the liquid is clear.
SECTION: Step7: AMPure beads clean up and library quality check
7.6. Carefully remove40 µLthe liquid/supernatant with 100 μL pipette tips and discard it, keep5 µLwithout disturbing the beads-pellet.
SECTION: Step7: AMPure beads clean up and library quality check
7.7. Wash the beads-pellet with175 µLfreshly prepared by gently dispensing it over the beads with 200 μL pipette tips. Let it rest for30 sand then remove the liquid.
SECTION: Step7: AMPure beads clean up and library quality check
7.8. Repeat washing .
SECTION: Step7: AMPure beads clean up and library quality check
7.9. Spin the tudes or plates in a minicentrifuge for30 sto collect all the residual liquid. 
SECTION: Step7: AMPure beads clean up and library quality check
7.10. Put the tubes or plates on a magnetic rack and remove the excess with 10 μL pipette tips.
SECTION: Step7: AMPure beads clean up and library quality check
7.11. Air dry for approximately30 sto evaporate the Ethanol.
SECTION: Step7: AMPure beads clean up and library quality check
7.12. Remove the tube or plate from the magnetic rack. Add16 µLnuclease free water and mix with a pipette (>10 times up and down) to resuspend the beads-pellet. Incubate for 2 min atRoom temperature.
SECTION: Step7: AMPure beads clean up and library quality check
7.13. Spin down for30 sif there is liquid on the wall of tubes or well, place the tube or plate back on the magnetic rack and wait for3 minuntil the liquid clears.
SECTION: Step7: AMPure beads clean up and library quality check
7.14. Transfer15 µLof the liquid/supernatant to a new, labeled Eppendorf tube or a new plate.
SECTION: Step7: AMPure beads clean up and library quality check
7.15. Qubit measurement of cleaned PCR products and pool 10 ng per sample to prepare a pooled library for library quality check with TapeStation following their standard protocols.
 

SECTION: Step4: Fragmentation
4.1. CRITICAL STEP: Pre-cooled (4°C) the Biorupter and holder at least30 min.
SECTION: Step4: Fragmentation
4.2. While waiting for cooling down, transfer genomic DNA or reverse-transcription products to a new, clean Bioruptor tubes and briefly spin down to make sure all the liquid is at the bottom of tubes.
SECTION: Step4: Fragmentation
4.3. Put samples on the ice for at least10 min.

SECTION: Step4: Fragmentation
4.4. CRITICAL STEP: Set the sonication parameter as following: 15s ON and 90s OFF, using 8 cycles.
SECTION: Step4: Fragmentation
4.5. CRITICAL STEP: Spin down the samples and carefully load the Bioruptor tubes to sonicater holder and close carefully, make sure there is no liquid at the side of tubes.
SECTION: Step4: Fragmentation
4.6. CRITICAL STEP: Put the tube holder into the sonication chamber and close the lids. 12 samples can be done each time. 
SECTION: Step4: Fragmentation
4.7. Spin down the tubes after shearing.
SECTION: Step4: Fragmentation
4.8. The fragmented product is ready for ligation.
 

SECTION: Step5: Ligation
5.1. Denature
SECTION: Step5: Ligation
5.2. According to the number of samples (Include extra 3 for pipetting errors), prepare an ET SSB dilution in a clean tube. In the table below is calculated for a whole plate of 96 wells (Master Mix per 100 samples): diluted ET SSB to 5 ng/µL (500 µg/mL in the stock solution) with Tris and then add same volume of Tris, for example 1 µL ET SSB in 100 µL tris buffer. Mix them, spin them down and place on ice. 
 
 
SECTION: Step6: Index PCR
6.1. Take the index primer (illumina i5 + i7) plate from the freezer to the fridge before the start.
SECTION: Step6: Index PCR
6.3. In a clean EP tube, transfer the needed volume of 10× AccuPrime buffer and AccuPrime DNA polymerase from the stock solution, gently mix, and spin down.
SECTION: Step5: Ligation
5.3.  Add20 µLof fragmented DNA to each well.
SECTION: Step5: Ligation
5.4. Add2 µLET SSB solution, pipette several times, and spin down.

SECTION: Step5: Ligation
5.5. Transfer to ThermoCycle and denature for 3 minat95 °C. 

SECTION: Step5: Ligation
5.6. Cooling down on ice immediately after heating and set on icefor at least5 min.

SECTION: Step5: Ligation
5.7.  Ligation
SECTION: Step5: Ligation
5.8. Spin down the heat denatured sample with a microcentrifuge.
SECTION: Step5: Ligation
5.9. According to the number of samples (Include extra 3 for pipetting errors), prepare a Master Mix containing per sample. In the table below is calculated for a whole plate of 96 wells (Master Mix per 100 samples). Mix them, spin them down and place on ice.
 

 

SECTION: Step5: Ligation
5.10. Add 28 µLligation master mix to the denatured samples, mix thoroughly with pipette, spin briefly.

SECTION: Step5: Ligation
5.11. Transfer to ThermoCycle for60 minligation at37 °C.

SECTION: Step5: Ligation
5.12. Purified with 1X AMPure XP beads (25 μL / 25 μL PCR reaction) and eluted with22 µLMiliQ H2O according to the purification procedures from Step7.
 

SECTION: Step6: Index PCR
6.2. According to the number of samples (include an extra 3 for pipetting errors), prepare a master mix containing per sample. The table below is calculated for a whole plate of 96 wells (Master Mix per 100 samples):
 

SECTION: Step6: Index PCR
6.4. Transfer2.9 µLMaster mix to each sample.
SECTION: Step6: Index PCR
6.7. Carefully place the PCR plate into the thermocycle machine and select the following program: 
 

SECTION: Step6: Index PCR
6.5. Transfer1 µLPrimer mix to each sample.
SECTION: Step6: Index PCR
6.6. Carefully mix them and spin them down.
SECTION: Step6: Index PCR
6.8. Purified with 0.8X AMPure XP beads (20 μL/25 μL PCR reaction) and eluted with22 µLMiliQ H2O according to the purification procedures from .
 



SECTION: Step3: Reverse transcription (RT)
3.1. Transfer 16 μL of extracted or heat-treated virome DNA/RNA to a clean PCR plate.