Automated RNA Library Prep | Transcriptome Sequencing of Arabidopsis Leaf Samples

Arabidopsis thaliana is the model species of plant molecular biology, and leaf transcriptome sequencing is a core technique for dissecting plant growth and development, stress responses and gene regulatory mechanisms — all of which depend on high-quality RNA libraries for accurate quantification. Conventional manual library construction is operationally complex, limited in throughput and prone to operator error that undermines reproducibility.

This application note presents an automated RNA library-construction workflow for Arabidopsis leaf samples on the MGISP-960 platform, combined with the Hieff NGS™ EvoMax RNA Library Prep Kit (dNTP, 12341ES). The method processes up to 96 samples per batch automatically, effectively eliminates manual error, and improves library-quality stability and experimental efficiency — providing standardized, high-efficiency library construction for large-scale Arabidopsis transcriptome studies.

Background and Pain Points

Compared with DNA workflows, automated RNA library preparation adds delicate magnetic-bead capture and reverse-transcription steps, which makes the manual route even harder to standardize:

•  mRNA capture and on-bead fragmentation involve delicate magnetic steps that are difficult to reproduce by hand.

•  The RNA workflow chains eight steps — capture, fragmentation, first- and second-strand cDNA synthesis, ligation, cleanup, PCR — multiplying manual-error opportunities.

•  Reverse-transcription and capture reagents are high-value; manual pipetting wastes expensive consumables.

•  Transcriptome cohorts of 96 samples exceed practical manual throughput (6–8 hours of hands-on time).

Application Scenarios

The automated RNA workflow supports the core questions of Arabidopsis molecular biology at 96-sample scale.

Figure 1. Application landscape covered by the automated Arabidopsis leaf RNA library-construction workflow.

The Automated Workflow

The protocol runs on the MGISP-960 with the EvoMax RNA Library Prep Kit (dNTP, 12341ES) and MGI-compatible UDB adapters. Manual touchpoints are limited to sample loading, adapter dilution (10 μM → 5 μM) and final library collection.

Figure 2. Automated Arabidopsis leaf RNA library-preparation workflow on the MGISP-960 (MGI library). 

Reagent List for the Experiment

Category	Cat.No.	Product name
RNA Library Preparation	12629ES	Hieff NGS™ mRNA Isolation Master Kit V2
	12341ES	Hieff NGS™ EvoMax RNA Library Prep Kit (dNTP)
Magnetic Beads	12601ES	Hieff NGS™ DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	13350ES	Hieff NGS™ Unique Dual Barcode Primer Kit for MGI, Set 1 (plate format)
Automation Consumables	83560ES / 83672ES / 83130ES	0.2 mL 96-well PCR plates (white, full skirt) / 96-deep-well plates (U-bottom, 1.3 mL) / 250 μL filtered sterile tips
User-Supplied Materials	—	Absolute Ethanol

Pre-Experiment Preparation

•  Equilibrate magnetic beads to room temperature for at least 30 minutes and resuspend fully before deck loading.

•  Prepare fresh 80% ethanol for all bead washes.

•  Dilute the adapter stock from 10 μM to 5 μM (manual pre-step, keep on ice).

•  Load one 96-sample consumable set and confirm deck layout, liquid volumes and tip integrity.

•  Assess total RNA integrity (RIN ≥ 7 recommended) before the run.

Experimental Tips

Sample & input

•  Start from intact total RNA: degraded RNA shifts the fragment distribution and reduces mRNA-capture efficiency.

•  Input 1,000 ng total RNA per sample and keep the mass consistent across the plate.

mRNA capture & fragmentation

•  Ensure beads are fully resuspended; 2× washes and complete Tris elution are the main levers of mRNA yield.

•  On-bead fragmentation at 94°C for 5 minutes in frag / primer buffer reproducibly yields ~400 bp inserts.

Adapter handling & PCR

•  Dilute adapters 10 μM → 5 μM manually before the run; 12 PCR cycles balance yield and duplication for 1,000 ng input.

•  For species or tissues with high rRNA background, switch to plant rRNA depletion (12254ES) instead of poly(A) capture.

Automation & QC

•  Run scripts exactly as specified in the 12341ES IFU — plate geometry and liquid classes are validated together.

•  Accept finished libraries at 20–40 ng/μL (Qubit) with a sharp ~400 bp peak (Qsep) before pooling.

Experimental Conditions and Results

 

Case results (Qubit 4.0):

All three libraries fell between 20 and 40 ng/μL with insert sizes of approximately 400 bp (Qsep), meeting sequencing-ready specifications for downstream transcriptome sequencing.

Figure 3. Library concentration across the validated Arabidopsis leaf RNA run (n = 3, 25 μL elution).

Figure 4. Arabidopsis leaf RNA library size distribution with a sharp single peak at ~400 bp.

Analysis of Experimental Results

Libraries were constructed from total RNA extracted from Arabidopsis thaliana leaf tissue using the RNA library‑construction workflow. Satisfactory library yield and well‑defined fragment‑size distribution were obtained, complying with standard quality‑control criteria for transcriptome sequencing.