Ligation - EecSeq

Manufacturer’s instructions

Adapter ligation

3.1 Dilute adapter stocks to the appropriate concentration, as outlined below:

Fragmented DNA	Adapter stock concentration	Adapter:insert molar ratio
1 μg	15 μM	10:1
500 ng	15 μM	20:1
250 ng	15 μM	40:1
100 ng	15 μM	100:1
50 ng	15 μM	200:1
25 ng	15 μM	200:1
10 ng	15 μM	200:1
5 ng	15 μM	200:1
2.5 ng	15 μM	200:1
1 ng	15 μM	200:1

For Puritz and Lotterhos (2017), a working stock of 40 μM was used, leading to a final adapter:insert molar ratio of ~ 50:1.

3.2 In the same plate/tube(s) in which end repair and A-tailing was performed, assemble each adapter ligation reaction as follows:

Component	Volume
End repair and A-tailing reaction product	30 μl
P1 Adapter stock (concentration as required) Barcode containing oligo	1.25 μl
P2 Adapter stock (concentration as required)	1.25 μl
PCR-grade water-	2.5 μl
Ligation Buffer-	15 μl
DNA ligase-	5 μl
Total Volume	55 μl

• Notes*
• Each P1 adapter has a unique barcode these barcodes can be combined with Illumina indices (added later via PCR) for high levels of multiplexing.
• The water, buffer and ligase enzyme should preferably be premixed and added in a single pipetting step. Premixes are stable for ≤24 hrs at room temperature, for ≤3 days at 4°C, and for ≤4 weeks at -20°C.

3.3 Mix thoroughly and centrifuge briefly.

3.4 Incubate at 20°C for 60 min. Note: to achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time—to a maximum of 4 hrs at 20°C, or overnight at 4°C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.

3.5 Proceed immediately to the next step.