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)

8.2 Fragmentation

1. Prepare 6.5 $\mu L$ of samples with ca 100 $ng$ of DNA (see previous chapter)
2. On ice, pipette up and down ULTRA II FS Reaction Buffer 10X, vortex 5" and spin
3. On ice, vortex 5" Ultra II FS Enzyme Mix and spin
4. 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
5. On ice, add 2.25 $\mu L$ of premix to each sample, vortex 5" and spin:

Component	Volume per library	Volume per plate
DNA	6.5 $\mu L$
Ultra II FS Enzyme Mix	0.5 $\mu L$	48 $\mu L$
Ultra II FS Reaction Buffer	1.75 $\mu L$	168 $\mu L$
Total	8.75 $\mu L$

6. 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
7. Optionally, put the NEBNext adaptor for Illumina out of the freezer (long to melt)
8. Optionally, samples can be stored overnight at $-{20}^{\circ }$

8.3 Adaptor ligation

1. 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)
2. 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
3. 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 $\mu L$

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

8.4 Size selection

1. 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)
2. Vortex PGTB beads from batch E at room temperature
3. 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
4. Add 3.5 $\mu L$ (~0.14X) of beads (336 $\mu L$ per plate) to each sample, mix, wait 5’ without the magnet
5. Place on magnet, wait 5" and discard ~35.5 $\mu L$ of supernatant
6. Add 100 $\mu L$ of fresh 80% Ethanol (9 600 $\mu L$ per plate) to the beads on the magnet, wait 30’ remove supernatant
7. 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
8. Air dry beads 3’ on magnet
9. 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’
10. Transfer 2 x 5 $\mu L$ of supernatant to 2 new plates
11. Optionally, samples can be stored overnight at $-{20}^{\circ }$

8.5 Enrichment and purification

protocol given for delivered oligos at $100mM$, not NEBNext tag at $10mM$

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

Component	Volume per library	Volume per plate/row/column
sample	5 $\mu L$
NEBNext Ultra II Q5 Master Mix	8.3 $\mu L$	796.8 $\mu L$
diluted Index Primer i5 (1:10)	1.6 $\mu L$	19.2 $\mu L$
diluted Index Primer i7 (1:10)	1.6 $\mu L$	12.8 $\mu L$
Total	16.5 $\mu L$

3. Thermocycle with following program
   • 30"@98°C
   • 8 cycles of 10"@98°C and 75"@65°C
   • 5'@65°C
   • Hold@4°C
4. Optionally, amplify only the first plate, assess it with electrophoresis, and adjust cycles number for the second amplifcation depending on gel migration
5. Pool PCR results (~16.5 $\mu L$ per sample) from the 2 plates into one (~33.3K $\mu L$ per sample)
6. Vortex PGTB beads from batch E at room temperature
7. 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)
8. Add 100 $\mu L$ of fresh 80% Ethanol (9 600 $\mu L$ per plate), wait 30’ remove supernatant
9. Repeat, add 100 $\mu L$ of fresh 80% Ethanol (9 600 $\mu L$ per plate), wait 30" remove supernatant
10. Air dry beads 3’ on magnet
11. 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
12. Place on magnet, wait 5’, transfer 22 $\mu L$ of supernatant to a new plate and store at $-{20}^{\circ }$