Chapter 8 Libraries preparation protocol

The protocol is given per sample with the corresponding volume for a plate of 96 samples in bracket.

8.1 Material preparation

0.5 \(\mu L\) of 10mM TrisHCL + 10 mM NaCl (48 \(\mu L\) per plate)
46.875 \(\mu L\) of 0.1X TE buffer (2 136.875 \(\mu L\) per plate) = 4.7 \(\mu L\) of TE buffer (213.7 \(\mu L\) per plate) + 42.3 \(\mu L\) of water (1 923.3 \(\mu L\) per plate)
400 \(\mu L\) of fresh 80% Ethanol (38 400 \(\mu L\) per plate)

Prepare 6.5 \(\mu L\) of samples with ca 100 \(ng\) of DNA (see previous chapter)
On ice, pipette up and down ULTRA II FS Reaction Buffer 10X, vortex 5" and spin
On ice, vortex 5" Ultra II FS Enzyme Mix and spin
On ice, premix 0.5 \(\mu L\) of Ultra II FS Enzyme Mix (48 \(\mu L\) per plate) with 1.75 \(\mu L\) of Ultra II FS Reaction Buffer (168 \(\mu L\) per plate), vortex and spin
On ice, add 2.25 \(\mu L\) of premix to each sample, vortex 5" and spin:

Thermocycle with following programs depending on electrophoresis quality:
- Good: 13'@37°C, 30'@65°C, Hold@-4°C
- Medium: 9'@37°C, 30'@65°C, Hold@-4°C
- Bad: 1'@37°C, 30'@65°C, Hold@-4°C
Optionally, put the NEBNext adaptor for Illumina out of the freezer (long to melt)
Optionally, samples can be stored overnight at \(-20^\circ\)

On ice, prepare diluted adaptor (1:5) with 0.125 \(\mu L\) of NEBNext adaptor for Illumina (12 \(\mu L\) per plate) diluted into 0.5 \(\mu L\) of 10mM TrisHCL + 10 mM NaCl (48 \(\mu L\) per plate)
On ice, premix 7.5 \(\mu L\) of NEBNext Ultra II Ligation Master Mix (720 \(\mu L\) per plate) with 0.25 \(\mu L\) of NEBNext ligation enhancer (24 \(\mu L\) per plate), vortex and spin
On ice, add 0.625 \(\mu L\) of diluted adaptor and 7.75 \(\mu L\) of premix to samples, mix and spin:

Component	Volume per library	Volume per plate
DNA	8.75 \(\mu L\)
NEBNext Ultra II Ligation Master Mix	7.5 \(\mu L\)	720 \(\mu L\)
NEBNext ligation enhancer	0.25 \(\mu L\)	24 \(\mu L\)
diluted NEBNext adaptor (1:5)	0.625 \(\mu L\)	50 \(\mu L\)
Total	17.25 \(\mu L\)

Incubate 15'@20°C with lid open
On ice, add 0.75 \(\mu L\) of USER Enzyme (72 \(\mu L\) per plate) to samples, mix and spin
Incubate 15'@37°C with lid hot (>47°C)
Optionally, samples can be stored overnight at \(-20^\circ\)

Bring the sample volume from 17.125 \(\mu L\) to 27.125 \(\mu L\) adding 10 \(\mu L\) of 0.1X TE buffer (1 716 \(\mu L\) per plate)
Vortex PGTB beads from batch E at room temperature
Add 7 \(\mu L\) (~0.28X) of beads (672 \(\mu L\) per plate) to each sample, mix, vortex 5" keeping beads, incubet 5’, spin, place on magnet, wait 5’, and transfer ~32 \(\mu L\) of sample to a new plate
Add 3.5 \(\mu L\) (~0.14X) of beads (336 \(\mu L\) per plate) to each sample, mix, wait 5’ without the magnet
Place on magnet, wait 5" and discard ~35.5 \(\mu L\) of supernatant
Add 100 \(\mu L\) of fresh 80% Ethanol (9 600 \(\mu L\) per plate) to the beads on the magnet, wait 30’ remove supernatant
Repeat, add 100 \(\mu L\) of fresh 80% Ethanol (9 600 \(\mu L\) per plate) to the beads on the magnet, wait 30" remove supernatant
Air dry beads 3’ on magnet
Remove magnet, elute into 12 \(\mu L\) of hot 0.1X TE (1 152 \(\mu L\) per plate) (~40°C), mix, incubate 2’, spin, put on magnet, wait 5’
Transfer 2 x 5 \(\mu L\) of supernatant to 2 new plates
Optionally, samples can be stored overnight at \(-20^\circ\)

protocol given for delivered oligos at \(100mM\), not NEBNext tag at \(10mM\)

Prepare diluted index (1:10) with 0.16 \(\mu L\) of Index Primer i5 and i7 diluted in 1.44 \(\mu L\) of mQ \(H_2O\) (1.92 \(\mu L\) of i5 per row and 1.28 \(\mu L\) of i7 per column)
Mix in each plate (2 for the 2 PCR), mix and spin :

Thermocycle with following program
- 30"@98°C
- 8 cycles of 10"@98°C and 75"@65°C
- 5'@65°C
- Hold@4°C
Optionally, amplify only the first plate, assess it with electrophoresis, and adjust cycles number for the second amplifcation depending on gel migration
Pool PCR results (~16.5 \(\mu L\) per sample) from the 2 plates into one (~33.3K \(\mu L\) per sample)
Vortex PGTB beads from batch E at room temperature
Add 30 \(\mu L\) (~0.9X) of beads (2 880 \(\mu L\) per plate), mix, vortex 5" keeping beads, spin, place on magnet, wait 5’, and remove supernatant (~ 63 \(\mu L\) per sample)
Add 100 \(\mu L\) of fresh 80% Ethanol (9 600 \(\mu L\) per plate), wait 30’ remove supernatant
Repeat, add 100 \(\mu L\) of fresh 80% Ethanol (9 600 \(\mu L\) per plate), wait 30" remove supernatant
Air dry beads 3’ on magnet
Remove magnet, elute into 22 \(\mu L\) of hot 0.1X TE (2 112 \(\mu L\) per plate) (~40°C), mix, incubate 2’, and spin
Place on magnet, wait 5’, transfer 22 \(\mu L\) of supernatant to a new plate and store at \(-20^\circ\)