TITLE: Metagenomic Library Prep from fecal sample lysate 
AUTHORS: Noah Noah Snyder-Mackler

[DESCRIPTION]
Metagenomic library prep from fecal sample lysate using Illumina DNA prep kit (1/2 reactions).

[GUIDELINES]
Abbreviations
BLT: Bead-Linked Transposomes
TB1: Tagmentation Buffer 1
MM: Master Mix
TWB: Tagmentation Wash Buffer
TSB: Tagmentation Stop Buffer
EPM: Enhanced PCR Mix
SPB: Sample Purification Beads
RSB: Resuspension Buffer

[STEPS]
SECTION: Preparation
1. Add 18 µL of nuclease-free water and 2 µL of sample to a PCR plate

SECTION: Tagmentation Reaction
2. Take out TB1 and keep on on ice. Turn on thermocycler to 37 °C 

SECTION: Tagmentation Reaction
3. Multiply by number of samples, for a 96 well plate use x100 and pipet mix together to create MM. Vortex BLT vigorously for 10 seconds to resuspend 
   Reagent x1x100BLT5 ul 500ulTB15 ul500ul

SECTION: Tagmentation Reaction
4. Vortex and add 10 µL of reaction MM to each well
 


SECTION: Tagmentation Reaction
5. Pipette mix slowly 5 times using multichannel (should have 30 µL of volume in each well
SECTION: Tagmentation Reaction
6. Cover and seal plate. Then run "TAG" on thermocycler
Choose the preheat lid option and set to 100 °C
Set the reaction volume to 30 µL
55 °C for 15 min 
Hold at 10 °C 
SECTION: Tagmentation Reaction
7. During TAG incubation take out TSB and place at 37 °C and vortex before addition to samples 

SECTION: Tagmentation Reaction
8. Add 10 µL of TSB to each sample well using repeater with 0.5 mL tip to stop enzymatic reaction 

 

SECTION: Tagmentation Reaction
9. Cover with thick plastic and run “PTC” protocol on thermocycler 
Choose the preheat lid option and set to 100 °C
Set the reaction volume to 40 µL
55 °C for 15 min 
Hold at 10 °C 
SECTION: Tagmentation Reaction
10. Place on magnetic stand until beads pellet and discard supernatant 
 

SECTION: Tagmentation Reaction
11. Wash 2x with 100 µLof TWB. Place on magnetic stand until beads pellet + remove and discard supernatant after each wash. After both washes add in another 100 µLof TWB to plate while on magnetic stand to prevent beads from drying while you prep PCR mix (don’t pipette mix)

SECTION: Make PCR Master Mix
12. Take out EPR and thaw on ice 
SECTION: Make PCR Master Mix
13. Make master mix, multiply by number of samples (use x100 for a 96 well plate) (use a 5 ml tube for a full plate of MM)
   Reagent x1x100EPR111,056Nuclease free water111,056

SECTION: Make PCR Master Mix
14. Place on magnetic stand until beads pellet + remove and discard supernatant (TWB)
SECTION: Make PCR Master Mix
15. Add 20 µL of MM using repeater and 5 mL attachment 

SECTION: Make PCR Master Mix
16. Add in 2.5 µL of i5 and i7 primers 

SECTION: Make PCR Master Mix
17. Pipette mix, cover with thick plastic and run “BLT” 12 cycle on thermocycler ( 60 min) 
Choose the preheat lid option and set to 100 °C
68 °C for 3 min
98 °C for 3 min
12 cycles of:
            98 °C for 45 s
            62 °C for 30 s
            68 °C for 2 min
68 °C for 1 min
Hold at 10 °C

 




SECTION: Bead Clean Up
18. Grab a new PCR plate and add 60 µL of nuclease-free water using a multichannel pipette and 45 µL of SPB using a repeater with a .5 ml tip to each sample well 

SECTION: Bead Clean Up
19. After PCR is complete spin down plate → place on magnetic stand → transfer 25 µL of supernatant to new PCR plate containing water and SPB. Discard old plate. 

SECTION: Bead Clean Up
20. Pipette mix 10 times and incubate for 5 min at Room temperature
During this incubation take out a new PCR plate and add 15 µL of SPB to each well using a repeater 
SECTION: Bead Clean Up
21. After the incubation place plate on magnetic stand until beads pellet 
SECTION: Bead Clean Up
22. Transfer 125 µL to the new plate containing the 15 µL of SPB. Discard old plate. 

SECTION: Bead Clean Up
23. Pipette mix 10 times and incubate for 5 min at Room temperature

SECTION: Bead Clean Up
24. Place on magnetic stand + remove and discard supernatant 
SECTION: Ethanol Washes
25. Keep plate on magnetic stand and prepare fresh 80% ethanol
SECTION: Ethanol Washes
26. While keeping the plate on the magnetic stand add 200 µLof 80% ethanol without mixing 

SECTION: Ethanol Washes
27. Incubate 30 s + remove and discard supernatant. Repeat ethanol wash and discard supernatant. 
 


SECTION: Ethanol Washes
28. Air dry while on magnetic stand (3 min)

 

SECTION: Elution
29. Remove beads from stand and add 32 µL of RSB to beads 

SECTION: Elution
30. Pipette mix to resuspend and incubate at Room temperature for 2 min

SECTION: Elution
31. Place on magnetic stand and transfer supernatant to new and final sturdy PCR plate. Seal, label, and store!