DNA Library Prep | Mouse Tissue(Skin and Heart) Samples | WGS

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12972ES	Hieff NGS^TM OnePot Pro DNA Library Prep Kit V4
Magnetic Beads	12601ES	Hieff NGS^TM DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	12330ES	Hieff NGS^TMStubby UDI Primer Kit for Illumina, Set4（1,152 Illumina Dual-Index Unique Adapters, Plate-Format, Set 4）
User-Supplied Materials	—	Absolute Ethanol

Pre-Experiment Preparation

1. Allow the magnetic beads to equilibrate to room temperature before use.

2. Prepare 80% ethanol.

3. Dilute the adapter 2-fold.

4. Prepare the sample according to the quantities listed in the table below.

No.	1	2	3	4	5	6	7	8	9	10	11	12
Type	Mouse skin sample							Mouse heart sample
Input Amount	30 ng	50 ng	30 ng	50 ng	30 ng	30 ng	50 ng	30 ng	30 ng	50 ng	50 ng	100 ng

Product description

Library Preparation Method	Automated library preparation: MGI SP-960
Input DNA	Sample input amount according to the table above(30~100 ng)
Fragmentation	4℃ 1 min, 35℃ 15 min, 72℃ 20 min, 4℃ hold
Adapter	Illumina UDI adapter, 2-fold dilution
Post-Ligation Cleanup & Size Selection	0.6× cleanup after ligation; 0.62×/0.15× Size selection
PCR Cycles	7 cycles
Post-PCR Cleanup	0.9× purification
Library Elution Volume	25 μL

Procedure

1. DNA Fragmentation / End Repair / dA-Tailing

Thaw and invert-mix all reagents in Table 1; keep on ice. Prepare the reaction mix on ice, mix gently (pipette or low-speed vortex), then spin briefly. Run the thermal program for fragmentation, end repair, and dA-tailing.

Table 1.DNA Fragmentation / End Repair / dA-Tailing

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Input DNA	X	Heated Lid: 105 °C	On
Smearase Buffer 4.0	10	4℃	1 min
Smearase Enzyme 4.0	10	35℃	15 min
ddH2O	Up to 60	72℃	20 min
-	-	4℃	Hold

2. Adapter Ligation
Adjust the Adapter concentration appropriately based on the Input DNA amount. For this experiment, dilute the UDI adapters 2-fold.

Table 2. Adapter Ligation Reaction

Name	Volume (μL)	Temperature	Time
dA-tailed DNA	60	-	-
Ligation Enhancer 4.0	30*	Heated Lid	Off
Rapid DNA Ligase 4.0	10	20℃	15 min
PE Adapter	5	4℃	Hold
ddH2O	Up to 110	-	-

[Note]: * Ligation Enhancer is viscous. Before use, invert and vortex thoroughly to mix completely, then briefly centrifuge.

3. Post Ligation Clean Up

This step uses magnetic beads to purify adapter-ligated products, removing unligated adapters and adapter dimers.

Purification is followed by size selection: first, perform a 0.6× cleanup and elute in 102 μL ddH₂O; then apply a 0.62×/0.15× double-sided size selection. Elute the final product in 20 μL for downstream amplification.

4. Library Amplification

This step performs PCR amplification to enrich the purified and size-selected adapter-ligated products. Prepare the reaction mixture and set the cycling program according to Table 3.

Table 3. Library Amplification Reaction

Name	Volume (μL)	Temperature	Time	Cycle Numbe
Adapter Ligated DNA	20	98℃	45 sec	1
2×Ultima HF Amplification Mix	25	98℃	15 sec	7
Primer Mix(12330ES)	5*	60℃	30 sec
Total	50	72℃	30 sec
-	-	72℃	1 min	1
-	-	4℃	Hold	-

5. Magnetic Bead Purification of Amplified Products
The product after amplification is purified using Hieff NGS^TM DNA Selection Beads (0.9×, Beads:DNA = 0.9:1).

Library Quality control results

No.	1	2	3	4	5	6	7	8	9	10	11	12
Type	Mouse skin sample							Mouse heart sample
Input Amount	30ng	50ng	30ng	50ng	30ng	30ng	50ng	30ng	30ng	50ng	50ng	100ng
Library Concentration (ng/μL)	32.4	54.2	61	85	75.6	75.6	92.2	31.6	31.2	43.6	43	63.2
Total Amount ng	810	1355	1525	2125	1890	1890	2305	790	780	1090	1075	1580
Qsep Main Peak	499bp	480bp	479bp	484bp	502bp	492bp	494bp	504bp	524bp	516bp	518bp	519bp

Library Qsep Electropherogram

Conclusion

Mouse skin and heart samples were processed using the enzymatic library preparation kit 12972ES on the automated workstation MGISP-960. The resulting libraries exhibited high yield and tight fragment size distribution, meeting all quality control standards.

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