LncRNA-Seq Library Preparation Protocol for Human/Mouse/Rat

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	12257ES	Hieff NGS™ MaxUp Human/Mouse/Rat rRNA Depletion Kit(rRNA ITS/ETS)
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	—	Kapa hyper RNA with riboerase HMR

Pre-Experiment Preparation

1. Equilibrate magnetic beads to room temperature before use.

2. Prepare 80% ethanol.

3. Sample Preparation: Mouse(Cell)

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

Kit → Species + Input (ng)		Yeasen 12629+12340		Kapa
		Library Conc.(ng/μL)	Library Conc.(nM, qPCR method)	Library Conc.(ng/μL)	Library Conc.(nM, qPCR method)
Mouse	200 ng	195 ng/μL	833.39 nM	23.8 ng/μL	81.05 nM
	500 ng	228 ng/μL	840.97 nM	46.6 ng/μL	173.82 nM

 
9. Sequencing Data Analysis

Sample Name	Clean Q20 (%)	Clean Q30 (%)	Clean GC (%)	rRNA (%)	Map Ratio (%)	Dup (%)	Gene Count (FPKM>0)	Transcript Count (FPKM>1)	Antisense (%)
25190R-02-01-200-Yeasen	99.68	98.11	49.06	0.67	98.00	20.62	83844	30497	98.6402
25190R-02-01-500-Yeasen	99.61	97.78	52.59	17.64	97.73	20.30	81731	29182	98.5857
25190R-02-01-200-KAPA	99.65	97.98	46.38	0.98	98.24	21.23	82434	31829	98.6442
25190R-02-01-500-KAPA	99.65	98.04	45.65	0.52	98.14	20.97	81956	31419	98.6345

[Note]: The high rRNA residue in the 500 ng sample may result from operational factors or other variables.

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 kapa kit.

2. Insert Size

Both kits generated libraries with distinct insert size distributions: the Yeasen kit yielded insert DNA primarily in the 200-250 bp range, while the Kapa kit produced inserts centered around 200 bp.

3. rRNA Depletion Efficiency:

KAPA: Consistently low rRNA levels (<1%) for both 200 ng and 500 ng inputs.

Yeasen: Excellent depletion at 200 ng (0.67%), but significantly higher rRNA contamination (17.64%) observed at 500 ng input. Operational errors or contamination factors are the suspected causes.

4. Gene Body Coverage

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

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