Sep 09, 2024

Public workspaceSingle_Stranded_Library_Workflow_Gut_Virome

Microbiome
DOI
dx.doi.org/10.17504/protocols.io.8epv5r5w5g1b/v1
  • 1University of Copenhagen
Xichuan Zhai
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DOI: dx.doi.org/10.17504/protocols.io.8epv5r5w5g1b/v1
Protocol Citation: Xichuan Zhai 2024. Single_Stranded_Library_Workflow_Gut_Virome. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r5w5g1b/v1
Manuscript citation:
https://doi.org/10.21203/rs.3.rs-4304844/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 08, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 107129
Keywords: Single-stranded library preparation, gut virome
Funders Acknowledgement:
VILLUM FONDEN
Grant ID: 23145
VILLUM FONDEN
Grant ID: 36242
Abstract
Protocol of single-stranded library (SSLR) preparation for virome study
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.
Materials
SM buffer: lab-prepared according to https://cshprotocols.cshlp.org/content/2006/1/pdb.rec8111.full?text_only=true
Pierce‱ Universal Nuclease for Cell Lysis: #88701
QIAamp Viral RNA Mini Kit (250): # 52906
SuperScript‱ IV VILO‱ Master Mix: #11756050
AMPure XP beads, 60 mL: #A63881
TE, pH 8.0, RNase-free: #AM9849
ET SSB (500 µg/mL): #M2401S
T4 Polynucleotide Kinase (10 U/μL): #EK0031
T4 DNA Ligase (5 U/μL): #EL0012
T4 DNA Ligase Buffer (10X) with 50% PEG-4000: #B69
Forward/reverse adapters: Lab designed and produced by IDT
AccuPrime‱ Taq DNA Polymerase System: #12339016
Nextera XT Index Kit v2 Sets: #FC-131-2001, 2002, 2003, 2004
Qubit 1X dsDNA HS Assay Kit: #Q33231
High Sensitivity D5000 ScreenTape Assay: #5067-5593
Protocol materials
ReagentFresh 80% Ethanol
Step 7.7
Safety warnings
Not applied
Ethics statement
Not applied
Before start
Fill bucket with crushed ice.
Step1: Virome isolation and extraction
Step1: Virome isolation and extraction
Checklist before starting
Note
Where: Fecal lab

Timing: 180 min

Reagents/kits
SM buffer: lab-prepared according to https://cshprotocols.cshlp.org/content/2006/1/pdb.rec8111.full?text_only=true
Pierce‱ Universal Nuclease for Cell Lysis: #88701
QIAamp Viral RNA Mini Kit (250): # 52906

Equipment
Centrifuge, 37°C incubator

Procedure

Make aliquots of Sample140 μL of enriched virome (procedure can be found from: dx.doi.org/10.17504/protocols.io.b2qaqdse).
Add Amount1 µL of 100-time diluted nuclease (check the stock for the dilution in SM buffer) to each sample and let them incubate at approximately Duration00:30:00 atTemperature37 °C per. sample. Vortex between each sample.
30m
Consider increasing the incubation time if you expect a lot of external DNA.
Immediately after addingAmount540 µL AVL buffer to inactivate nucleases.
Mix the mixture by pulse vortexing.
Incubation
Mix
Incubate at TemperatureRoom temperature for Duration00:10:00 .
10m
Incubation
Change gloves.
Briefly centrifuge the mixture with a microcentrifuge.
AddAmount560 µL of absolute ethanol (96%) to the sample mixture.
Mix
CRITICAL STEP: Mix very well by pulse-vortex.
Critical
Briefly centrifuge the mixture with a microcentrifuge.
AddAmount630 µL of the sample mixture to the spin column.
CRITICAL STEP: Do not touch the column rim with the pipet.
Centrifuge at Centrifigation6,000 x g, Room temperature change the collection tube and repeat steps 1.12 to load all the extracts.
Centrifigation
AddAmount500 µL AW1 buffer to the spin column the following:

Wash
Centrifuge atCentrifigation6,000 x g, Room temperature, 00:01:00 , change the collection tube.
1m
Centrifigation
Add Amount500 µL AW2 buffer to the spin column.
Wash
Centrifuge atCentrifigation20,000 x g, Room temperature, 00:03:00 , change the collection tube.
3m
Centrifigation
Centrifuge at Centrifigation20,000 x g, Room temperature, 00:01:00 . Place the spin column in a low-binding RNAse-free 1.5 mL tube.
1m
Centrifigation
Add Amount30 µL of AVE (elution buffer) to the spin column and incubate TemperatureRoom temperature forDuration00:01:00 .
1m
Incubation
CRITICAL STEP: Pipet the AVE buffer directly onto the filter membrane without touching it with the pipet tip.
Critical
Centrifuge atCentrifigation6,000 x g, Room temperature, 00:01:00 to collect the filtrates.
Note
PAUSE POINT Send for next step or store viral DNA/RNA at -80°C.

CAUTION
NOT interested in RNA??? THEN GO Go togo to step #4 .
Interested in DNA and ssRNA, THEN GO Go togo to step #3 .
Interested in DNA, dsRNA and ssRNA, THEN GO Go togo to step #2 .
Remember to include positive (Mock from extraction) and Negative (H2O from extraction) controls for each extraction.

1m
Centrifigation
Step2: Heat treatment
Step2: Heat treatment
Checklist before starting
Note
Where: Clean-lab

Timing: 3 min

Reagents
No reagents needed

Equipment
UV-beach, Thermocycler, Microcentrifuge

Procedure

Preheated ThermoCycle machine to Temperature95 °C and sterilized PCR tubes or plates depending on the how many samples are used for reverse transcription.
AddAmount16 µL extracted virome in PCR tubes or platesand prepare on ice.
Briefly centrifuge the mixture with a microcentrifuge.
Put tubes or plates for heat treatmentTemperature95 °C forDuration00:03:00 .
3m
Incubation
CRITICAL STEP: Transfer samples to ice immediately afterDuration00:03:00 .

3m
Critical
PAUSE POINT Ready for reverse transcription.
Note
CAUTION
If you would like to look at the RNA in your samples, it is better to finish extraction, heat treatment, and reverse transcription on the same day.
Pipette, filtered pipette tips, and workbench need to be sterilized by UV lamp for 20 min prior work.
Remember to include Positive (Mock from extraction Step1) and Negative (SM buffer from extraction) controls for the reaction.

Pause
Step3: Reverse transcription (RT)
Step3: Reverse transcription (RT)
Checklist before starting
Note
Where: Clean-lab

Timing: 25 mim

Reagents/kits
SuperScript‱ IV VILO‱ Master Mix: #11756050
AMPure XP beads, 60 mL: #A63881
TE, pH 8.0, RNase-free: #AM9849

Equipment
Thermocycler, Microcentrifuge, UV workbench

Procedure

Transfer 16 μL of extracted or heat-treated virome DNA/RNA to a clean PCR plate.
Add Amount4 µL SuperScript‱ IV VILO‱ Master Mix to each sample and mix thoroughly, spin them down.
Carefully place the PCR plate into the ThermoCycle machine and select the following program:
AB
Temperature profile
25°C10 min
50°C10 min
85°C5 min
4°C∞
PCR
Purified with 1X AMPure XP beads (25 μL / 25 μL PCR reaction) and eluted withAmount20 µL Tris
buffer (10 mM, pH8.0) according to the purification procedures fromGo togo to step #7 .
Note
PAUSE POINT Samples can be stored in the fridge or freezer for downstream preparations.

Pause
Step4: Fragmentation
Step4: Fragmentation
Checklist before starting
Note
Where: Basement (using ID card and key for access)

Timing: 15 min

Reagents
No reagent needed

Equipment
Bioruptor‱ Pico sonication device (#B01060010), Microcentrifuge

Procedure

CRITICAL STEP: Pre-cooled (4°C) the Biorupter and holder at leastDuration00:30:00 .
30m
Critical
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.
Put samples on the ice for at leastDuration00:10:00 .

10m
CRITICAL STEP: Set the sonication parameter as following: 15s ON and 90s OFF, using 8 cycles.
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.
CRITICAL STEP: Put the tube holder into the sonication chamber and close the lids. 12 samples can be done each time.
Spin down the tubes after shearing.
The fragmented product is ready for ligation.
Note
PAUSE POINT Samples can be stored in fridge or freezer for downstream preparations.
CAUTION
Pregnant women should not stay away from the running machine.
DO NOT turn on the instrument without water.
Distilled water should be used to fill the tank.
Always keep 12 tubes for each run to ensure successful shearing.

Step5: Ligation
Step5: Ligation
Checklist before starting
Note
Where: Clean-lab
Timing: 60 min

Reagents
ET SSB (500 µg/mL): #M2401S
T4 Polynucleotide Kinase (10 U/μL): #EK0031
T4 DNA Ligase (5 U/μL): #EL0012
T4 DNA Ligase Buffer (10X) with 50% PEG-4000: #B69
Forward/reverse adapters: Lab designed and produced by IDT
AMPure XP beads, 60 mL: #A63881

Equipment
Thermocycler, Microcentrifuge

Procedure

Denature
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.
ABC
ReagentPer 1 sample µL Per 100 samples µL
Fragmented DNA 20 -
ET SSB dilution (10 ng) 2 100
Total denaturation reaction volume 22
AddAmount20 µL of fragmented DNA to each well.
AddAmount2 µL ET SSB solution, pipette several times, and spin down.

Transfer to ThermoCycle and denature for Duration00:03:00 atTemperature95 °C .

3m
Cooling down on ice immediately after heating and set TemperatureOn ice for at leastDuration00:05:00 .

5m
Ligation
Spin down the heat denatured sample with a microcentrifuge.
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.
ABC
95 ℃ for 3min, cool down on ice immediately for at least 5 min
ReagentsPer 1 sample µL Per 100 samples µL
PEG-4000 (50%)101000
H2O8800
T4 ligase buffer (10X final)5500
T4 PNK (10,000 units/mL)1100
Add the above reagents one by one and then mix for the 1st time
Forward adapter (1 pmol)1 100
Reserve adapter (1 pmol)1 100
Add the above adapters one by one and then mix for the 2nd time
T4 DNA ligase (400,000 units/mL)2 200
Add the above reagents one by one and then by finger flicking to mix
Final volume5050000
37 ℃ for 60 min (set the lid-heating off)

Note
CRITICAL STEP for prepare master mix:

Make sure the T4 ligase buffer and PEG are dissolve and mix thoroughly;
Add the needed volume of PEG-4000 (sticky, do it slowly), ligase buffer and PNK first and mix thoroughly, then add adapters and mix again, ligase should be added lastly during cooling down the samples and mix thoroughly without vortexing.



Add Amount28 µL ligation master mix to the denatured samples, mix thoroughly with pipette, spin briefly.

Transfer to ThermoCycle forDuration01:00:00 ligation atTemperature37 °C .

1h
Purified with 1X AMPure XP beads (25 μL / 25 μL PCR reaction) and eluted withAmount22 µL MiliQ H2O according to the purification procedures from Step7.
Note
PAUSE POINT Store in fridge for short-time or freezer until beads clean-up.

CAUTION
Ensure that you have booked a ThermoCycle and preheated it toTemperature95 °C .
Pipette, filtered pipette tips, and workbench need to be sterilized by UV lamp forDuration00:20:00 prior work.
During preparation, reagents and PCR plate must be on ice/cold block. Reagents are stored at Temperature-20 °C .
Spin down reagents before using them. Avoid vortexing enzymes.
Remember to include a Positive (Mock from extraction), Negative (H2O from extraction) & Blank (H2O for the ligation) controls for the reaction.

Step6: Index PCR
Step6: Index PCR
Checklist before starting
Note
Where: Seq-lab

Timing: 30 mim

Reagents
AccuPrime‱ Taq DNA Polymerase System: #12339016
Nextera XT Index Kit v2 Sets: #FC-131-2001, 2002, 2003, 2004

Equipment
Thermocycler, Minicentrifuge

Procedure

Take the index primer (illumina i5 + i7) plate from the freezer to the fridge before the start.
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):
ABC
Per 1 sample μL Per 100 samples μL
Purified ligated DNA21.1
10× AccuPrime buffer2.5250
AccuPrime DNA polymerase0.440
Primer P5 (i5)1
Primer P7 (i7)
Final volume25

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.
TransferAmount2.9 µL Master mix to each sample.
TransferAmount1 µL Primer mix to each sample.
Carefully mix them and spin them down.
Carefully place the PCR plate into the thermocycle machine and select the following program:
ABC
Temperature profile
95°C2min
95°C15sx 20 cycles
57°C30s
68°C30s
4°C∞

PCR
Purified with 0.8X AMPure XP beads (20 μL/25 μL PCR reaction) and eluted withAmount22 µL MiliQ H2O according to the purification procedures fromGo togo to step #7 .
Note
PAUSE POINT Store in fridge for short-time or freezer until beads clean-up.

CAUTION
During the preparation of index PCR, reagents and PCR plate must be on ice/cold block. Lead opening while on cold block. Reagents are stored at -20°C.
Gently mix and spin down reagents before using them. Avoid vortexing enzymes.
WARNIN!!! Change pipette tips and PCR leads during work process!



Pause
Step7: AMPure beads clean up and library quality check
Step7: AMPure beads clean up and library quality check
27m
27m
Checklist before starting
Note
Where: Seq-lab

Timing: 30 min
Reagents
AMPure XP beads, 60 mL: #A63881
Qubit 1X dsDNA HS Assay Kit: #Q33231
High Sensitivity D5000 ScreenTape Assay: #5067-5593

Equipments/kits
HulaMixture, Invitrogen‱ Qubit‱ 4 Fluorometer, TapeSation4200 with high-sensitive D5000 Screen Tape (#5067-5592)

Procedure

Place AMPure XP beads into the Hula mixer to resuspend the beads and to equilibrate toTemperatureRoom temperature forDuration00:15:00 .
15m
Label an Eppendorf tube or PCR plate with your sample ID and transfer the Amount25 µL PCR product.
TransferAmount20 µL (!!! 0.8X) of Beads solution to each PCR product (Amount25 µL ) and mix with 100 μL pipette tips (10 times up and down), resulting in aAmount45 µL mixture.
Incubate for Duration00:05:00 atTemperatureRoom temperature .
5m
Incubation
Place the tube or plate with the mixture into the magnetic rack for 2-4 min, until the liquid is clear.
Carefully removeAmount40 µL the liquid/supernatant with 100 μL pipette tips and discard it, keepAmount5 µL without disturbing the beads-pellet.
Wash the beads-pellet withAmount175 µL freshly preparedReagentFresh 80% Ethanol Contributed by users by gently dispensing it over the beads with 200 μL pipette tips. Let it rest forDuration00:00:30 and then remove the liquid.
30s
Repeat washingGo togo to step #7.7 .
Spin the tudes or plates in a minicentrifuge forDuration00:00:30 to collect all the residual liquid.
30s
Put the tubes or plates on a magnetic rack and remove the excess with 10 μL pipette tips.
Air dry for approximatelyDuration00:00:30 to evaporate the Ethanol.
30s
Remove the tube or plate from the magnetic rack. AddAmount16 µL nuclease free water and mix with a pipette (>10 times up and down) to resuspend the beads-pellet. Incubate for Duration00:02:00 atTemperatureRoom temperature .
2m
Spin down forDuration00:00:30 if there is liquid on the wall of tubes or well, place the tube or plate back on the magnetic rack and wait forDuration00:03:00 until the liquid clears.
3m 30s
TransferAmount15 µL of the liquid/supernatant to a new, labeled Eppendorf tube or a new plate.
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.
Note
PAUSE POINT Send for sequencing or store in the fridgeTemperature4 °C for short time or freezer Temperature-80 °C for long time.


Pause