LncRNA-Seq Library Preparation Protocol for Plant

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12340ES	Hieff NGS™ EvoMax RNA Library Prep Kit (Strand-specific)
rRNA Depletion	12254ES	Hieff NGS™ MaxUp rRNA Depletion Kit (Plant)
Magnetic Beads	12601ES	Hieff NGSTM DNA selection Beads (Superior Ampure XP alternative)
	12602ES	Hieff NGS™ RNA Cleaner
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	—	Qiagen Fastselect plant rRNA depl+NEB ultra II directional kit

Pre-Experiment Preparation

1. Equilibrate magnetic beads to room temperature before use.

2. Prepare 80% ethanol.

3. Sample Preparation: Arabidopsis(Leaves), wheat(Stem internode), Soybean(Soybean leaf)

Product description

Procedure

1. rRNA Depletion and RNA Fragmentation Protocol

1). Sample Requirements

Input Material: 100 ng – 1 μg Total RNA (Volume ≤ 10 μL).

Sample Type: Compatible with Human, Mouse, or Rat sources. Suitable for both intact RNA and degraded RNA (e.g., FFPE).

2). Procedure

Step 1: Probe Hybridization

Prepare Mix: In a PCR tube, combine:

Table 1. Probe Hybridization Reaction Setup and Cycling Conditions

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Hybridization Buffer	3	Heated Lid: 105 °C	On
		95℃	2 min
Human Probe Mix (rRNA & ITS/ETS)	2	95℃-42℃	0.1℃/s
Total RNA	10 (100 ng~1 μg)	42℃	5 min
Total	15	4℃	Hold

Step 2: RNase H Digestion

Table 2. RNase H Digestion Reaction Setup and Cycling Conditions

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
RNase H Buffer	3	Heated Lid: 75 °C	On
RNase H	2	65℃	30 min
Product from the previous step	15	4℃	Hold
Total	20	-	-

Step 3: DNase I Digestion

Table 3. DNase I Digestion Reaction Setup and Cycling Conditions

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
DNase I Buffer	27.5	Heated Lid: 50 °C	On
DNase I	2.5	37℃	30 min
Product from the previous step	20	4℃	Hold
Total	50	-	-

Step 4: RNA Purification & Fragmentation

1)Bind: Add 110 μL RNA Cleaner Beads (2.2× ratio) to the 50 μL reaction. Mix and incubate at room temperature for 5 min.

2)Wash: Separate on a magnetic stand, discard supernatant, and wash twice with 200 μL fresh 80% Ethanol.

3)Dry: Air-dry beads for 5–10 min.

4)Elute & Fragment:

Remove from magnet and resuspend beads in 18.5 μL Frag/Prime Buffer. Incubate at room temperature for 5 min.

Separate on magnet and transfer 17 μL supernatant to a new tube.

5)Fragment: Incubate the eluted RNA at the appropriate temperature (refer to kit guidelines based on RNA quality).

Table 4. Recommended RNA 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 5. 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 °C	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 6. 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 °C	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 7. 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 8. 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

Species	Input	Tissue	Yeasen Qubit (ng/μL)	Qiagen Qubit (ng/μL)	Yeasen Yield (nM)	Qiagen Yield (nM)
Arabidopsis	200 ng	Leaves	9.94	N/A	38.01	18.91
	500 ng	Leaves	36.00	N/A	162.35	34.07
Wheat	200 ng	Stem	12.40	N/A	57.55	7.69
	500 ng	Stem	32.00	N/A	147.10	15.70
Soybean	200 ng	Leaf	14.70	N/A	59.41	9.78
	500 ng	Leaf	45.40	N/A	217.90	32.92

9. Sequencing Data Analysis

Sample	Raw Q20 (%)	Raw Q30 (%)	Clean GC (%)	rRNA (%)	Map Ratio  (%)	Dup (%)	Transcript Count  (FPKM>0)	Transcript Count (FPKM>1)	Antisense (%)
Arabidopsis-200 ng-Yeasen	99.3047	97.5073	43.3947	0.28	99.67	72.447	29349	12020	99.6957
Arabidopsis-500-Yeasen	99.2825	97.4239	43.3926	0.38	99.61	71.0499	29749	12517	99.6651
wheat-200 ng-Yeasen	98.9258	96.2589	41.6786	0.25	99.63	65.9915	33288	14873	98.8341
wheat-500 ng-Yeasen	98.6908	95.7754	41.5323	0.28	99.65	65.2823	33403	14874	98.8235
Soybean-200 ng-Yeasen	98.9963	96.5581	57.9269	1.46	97.74	27.1055	50577	26646	99.0205
Soybean-500 ng-Yeasen	99.0684	96.7085	57.07	0.39	97.65	27.7292	51012	27307	99.0619
Arabidopsis-200-NEB	98.6132	95.5193	50.1966	2.93	97.63	40.756	48644	26521	99.0419
Arabidopsis-500-NEB	98.4541	95.2063	50.3922	3.77	97.65	33.4618	49299	26660	99.0922
wheat-200-NEB	99.3093	97.5129	42.6393	0.34	99.37	71.6619	44743	15595	98.6837
wheat-500-NEB	99.321	97.5252	42.503	0.21	99.26	69.574	45879	16483	98.7744
Soybean-200-NEB	98.8402	96.071	41.159	2.38	98.72	62.7176	47611	20598	98.0095
Soybean-500-NEB	98.8072	95.9819	41.3857	3.21	98.76	60.5638	47340	20438	98.0595

RNA-seq Insert Size Analysis                             

Gene Body Coverage 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 200-250 bp.

3. rRNA Depletion Efficiency:

Yeasen demonstrated consistently superior depletion (<1.5% rRNA) across all species and inputs. NEB showed species-dependent variability, with high efficiency in wheat (<0.35%) but elevated rRNA levels in Arabidopsis and Soybean (up to 3.77%).

4. Strand Specificity

Both kits maintained excellent strand specificity. Yeasen showed consistently higher antisense ratios (>98.8%) compared to NEB (>98.0%) across all samples.