mRNA-Seq Library Preparation Protocol for Human, Mouse, and Plant

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12340ES	Hieff NGS™ EvoMax RNA Library Prep Kit (Strand-specific)
mRNA Isolation	12629ES	Hieff NGS™ mRNA Isolation Master Kit V2
Magnetic Beads	12601ES	Hieff NGSTM DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	12330ES	Hieff NGSTM Stubby UDI Primer Kit for Illumina, Set4（1,152 Illumina Dual-Index Unique Adapters, Plate-Format, Set 4）
User-Supplied Materials	—	Absolute Ethanol
Control Reagent	—	NEB ultra II directional kit with Poly A selection

Pre-Experiment Preparation

1. Equilibrate magnetic beads to room temperature before use.

2. Prepare 80% ethanol.

3. Sample Preparation: human(pure T cells or mixed cells), mouse(brain), plant(wheat, Stem internode)

Product description

Procedure

1. mRNA Purification and Fragmentation

This protocol describes the enrichment of poly(A)+ mRNA from high-quality eukaryotic total RNA (Animals, Plants, Fungi) using Hieff NGS^TM mRNA Isolation Master Kit V2 (Yeasen Cat#12629).

• Input Material: 10 ng – 4 μg Total RNA (RIN > 7). In this experiment, Input RNA 200 ng, 500 ng.
• Volume: Adjust RNA volume to ≤50 μL with Nuclease-Free Water.
• Note: Not suitable for FFPE samples or RNA lacking poly(A) tails.

1. Bind mRNA

Equilibrate beads to room temperature (~30 min).

Mix 50 μL Total RNA (10 ng–4 μg) + 50 μL Beads. Pipette 6×.

Incubate (Thermal Cycler): 65°C for 5 min, 25°C for 5 min, Hold at 25°C

2. Wash & Elute

Separate on magnet (5 min). Discard supernatant.

Wash with 200 μL Wash Buffer. Pipette 6×. Separate on magnet. Discard supernatant.

Repeat wash step once (Total 2 washes).

Elute by resuspending in 50 μL Tris Buffer. Pipette 6×.

Incubate (Thermal Cycler): 80°C for 2 min, Hold at 25°C

3. Re-bind

Add 50 μL Binding Buffer. Pipette 6×. Incubate at Room Temperature for 5 min.

Separate on magnet (5 min). Discard supernatant.

Wash with 200 μL Wash Buffer. Pipette 6×. Separate on magnet. Discard supernatant completely.

4. Fragment

Resuspend beads in 18.5 μL Frag/Prime Buffer. Pipette 6×.

Incubate in Thermal Cycler (Pre-heated to 94°C) based on desired size. In this experiment, the RNA was fragmented at 94°C for 7 minutes.

Table 1. Recommended mRNA Fragmentation Procedure

Separate immediately on magnet. Transfer 17 μL supernatant to a new tube. 

Proceed to First-Strand cDNA Synthesis.

2. 1^st cDNA Strand Synthesis

This step synthesizes first-strand cDNA from enriched/fragmented target RNA (prepared via Poly(A) selection or rRNA depletion). 

Reagent Preparation: Thaw the 1^st strand synthesis reagents on ice, mix well by pipetting.

Reaction Setup: Assemble the reaction mixture in a PCR tube and perform the cDNA synthesis reaction.

Table 2. cDNA synthesis reaction system and Procedure

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Frag/Prime Buffer with Fragmented RNA	17	Heated Lid: 105 ℃	On
		25℃	10 min
1st Reaction Module 2.0	8	42℃	15 min
Total	25	70℃	15 min
-	-	4℃	Hold

3. 2^nd cDNA Fragmentation / End Repair / dA-Tailing

Thaw all reagents listed in Table 1, invert to mix thoroughly, and keep on ice. On ice, prepare the reaction mixture as specified in Table 1. Gently pipette up and down or use low-speed vortexing to mix, then briefly centrifuge to collect the reaction liquid at the bottom of the tube.

Table 3. PCR Reaction for DNA Fragmentation / End Repair / dA-Tailing

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
1st Strand cDNA	25	Heated Lid: 105 ℃	On
2nd Reaction Module (dUTP)*	35	16℃	30 min
Total	60	72℃	15 min
-	-	4℃	Hold

4. Adapter Ligation

The adapter should be diluted to an appropriate concentration based on the input DNA amount. Thaw all reagents listed in Table 4, invert to mix thoroughly, and keep on ice. On ice, prepare the reaction mixture as specified in Table 4. Perform the adapter ligation reaction according to the program in Table 4.

Table 4. Adapter Ligation Reaction

Name	Volume (μL)	Temperature	Time
dA-tailed DNA	60	Heated Lid	Off
Ligation Reaction Module	35	20℃	15 min
PE Adapter	5*(3 μM)	4℃	Hold
ddH2O	Up to 100	-	-

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

5. Post Ligation Clean Up

This step uses magnetic beads to purify the adapter-ligated products. Purification removes unligated adapters or adapter dimers and other ineffective byproducts.

In this experiment, a post-ligation cleanup followed by size selection was used: perform 0.6× cleanup on the ligation product, elute with 102 μL ddH₂O, then carry out size selection at a ratio of 0.6×/0.2×. The final product was eluted in 20 μL for the next amplification step.

6. 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

7. Magnetic Bead Purification of Amplified Products

The amplified products were purified using Hieff NGS^TM DNA Selection Beads (0.9×, Beads:DNA = 0.9:1).

8. Library Quality Control

Kit → Species + Input (ng)		Yeasen 12629+12340		NEB
		Library Conc.(ng/μL)	Library Conc.(nM, qPCR method)	Library Conc.(ng/μL)	Library Conc.(nM, qPCR method)
Human	200 ng	38.4	179.75 nM	2.6 ng/μL	11.72 nM
	500 ng	92.8 ng/μL	453.98 nM	7.04 ng/μL	31.29 nM
Mouse	200 ng	60.6 ng/μL	350.91 nM	5.56 ng/μL	25.49 nM
	500 ng	120 ng/μL	639.97 nM	8.52 ng/μL	37.61 nM
Wheat	200 ng	16.2 ng/μL	75.07 nM	0.85 ng/μL	2.9 nM
	500 ng	38.2 ng/μL	180.91 nM	2.9 ng/μL	15.1 nM

RNA-seq Insert Size Analysis

Gene Body Coverage Analysis

Strand Specificity Analysis

Analysis of Experimental Results

1. Library Yield

Yeasen demonstrated superior performance in library yield, with both Qubit (ng/μL) and qPCR (nM) quantification results being significantly higher than those of the NEB kit.

2. Insert Size

Both kits produced comparable insert sizes, with a distribution centered around 250-300 bp.

3. Gene Body Coverage

Yeasen Kit: Showed superior coverage at the 5' end of the transcripts.

NEB Kit: Exhibited stronger coverage intensity at the 3' end.

4. Strandedness

The Yeasen kit demonstrated better strand specificity than the NEB kit at both input amounts tested (200 ng and 500 ng).