GenieSeq Universal RNA-seq Library Prep Kit (Illumina & MGI, Plate) (MORV0077)

Components

Reagents are supplied prepackaged in a 96-well plate. Columns 10–12 are vacant and can be used to prepare reaction mixes or reagents such as magnetic beads and adapters.

Storage

Store at -30 ~ -15°C.

You will also need: adapters with matching PCR primers (stubby unique dual index sets for Illumina or MGI); GeniePure RNA Clean-up Beads and GeniePure DNA Clean-up Beads; an mRNA enrichment or rRNA depletion kit; RNA QC reagents such as the GenieQuant RNA HS Assay Kit or GenieQuant RNA BR Assay Kit; the GenieQuant 1× dsDNA HS Assay Kit for library QC; freshly prepared 80% ethanol; nuclease-free water, tubes and filter tips; a PCR instrument, magnetic rack, fluorometer and Agilent 2100 Bioanalyzer or equivalent.

1. Enrich and fragment RNA
   • Option A, poly(A) capture (10 ng - 4 μg intact eukaryotic total RNA in 50 μl): capture mRNA with poly(A) capture beads over two rounds of binding (65°C 5 min, 25°C 5 min, 4°C hold), washing and elution (80°C 2 min). Resuspend the final beads in 18 μl Frag/Prime Buffer 2, fragment at 85°C for 6 min (4°C hold), then transfer 16 μl supernatant to a new tube.
   • Option B, rRNA depletion (10 ng - 1 μg total RNA): deplete rRNA following the depletion kit instructions. Purify with 110 μl (2.2×) RNA clean-up beads (15 min on ice), wash twice with 200 μl 80% ethanol and air-dry for 5–10 min. Elute in 18 μl Frag/Prime Buffer 2 (room temperature, 2 min), transfer 16 μl supernatant and fragment at 85°C for 6 min (4°C hold).
   • Option C, purified mRNA or total RNA (0.5–100 ng): mix 8 μl RNA with 8 μl 2× fragmentation/priming buffer and fragment at 85°C for 6 min (4°C hold).
   • Do not pause between fragmentation and first-strand synthesis, as mRNA degrades easily. See Table 1 for other fragmentation conditions.
2. Synthesise first-strand cDNA
   • Mix 16 μl fragmented mRNA with 9 μl 1st Synthetic Master Mix 2 (25 μl total). Protect the master mix from light.
   • Run (heated lid 105°C): 25°C 10 min; 42°C 15 min; 70°C 15 min; 4°C hold. Proceed immediately.
3. Synthesise second-strand cDNA with end repair and dA-tailing
   • Add 40 μl 2nd Synthetic Master Mix 2 to the 25 μl first-strand product (65 μl total). Use the dNTP mix for non-strand-specific libraries or the dUTP mix for strand-specific libraries.
   • Run (heated lid 105°C): 16°C 30 min; 65°C 15 min; 4°C hold. Products can be stored at -30 ~ -15°C for 24 h.
4. Ligate adapters
   • Add 5 μl adapter to the 65 μl ds cDNA and mix, then add 30 μl Ligase Master Mix 2 (100 μl total). Dilute the adapter according to RNA input:

• Run (heated lid 105°C): 20°C 15 min; 4°C hold. Ligation products can be stored at 2 ~ 8°C for 1 h.

5. Purify or size-select
   • Purification (broad library size, inserts >200 bp): add 60 μl (0.6×) DNA clean-up beads, incubate at room temperature for 10 min, wash twice with 200 μl 80% ethanol, air-dry for 5–10 min, elute in 22 μl nuclease-free water and transfer 20 μl.
   • Size selection: purify with 60 μl (0.6×) beads as above but elute in 102 μl and transfer 100 μl. Add 60 μl (0.6×) beads, incubate 10 min and transfer 150 μl supernatant to a new tube. Add 10 μl (0.1×) beads, incubate 10 min, wash twice with 80% ethanol, air-dry, elute in 22 μl and transfer 20 μl. These ratios give a 280–380 bp Illumina insert after fragmentation at 85°C for 6 min; see Table 1 for other sizes.
   • Do not carry over beads. Purified products can be stored at -30 ~ -15°C for 24 h.
6. Amplify the library
   • Mix 20 μl purified ligation product, 5 μl PCR Primer Mix 6 for Illumina/MGI (supplied with the adapter kit) and 25 μl HiFi Amplification Mix 3 (50 μl total).

Recommended PCR cycles:

• Purify the PCR product with 45 μl (0.9×) DNA clean-up beads (10 min at room temperature). Optionally, to remove small fragments, resuspend the beads in 50 μl of a small-fragment removal buffer for 5 min before the ethanol washes. Wash twice with 200 μl 80% ethanol, air-dry for 5–10 min, elute in 25 μl nuclease-free water and transfer 23 μl.

7. Check library quality
   • Analyse size distribution with an Agilent DNA 1000 kit. A peak at 90–140 bp indicates adapter dimers: dilute the library to 50 μl with nuclease-free water and repeat the 0.9× bead purification.

Table 1. Size selection conditions for ligation products (stubby adapters)

Bead ratios are relative to the initial DNA volume (100 μl). Final library length depends on the bead volumes used.

Q1: How can I improve a low library concentration?

• Library yield depends on RNA integrity and input. If high-quality RNA is not available, increase total RNA input to up to 1 μg, or prepare several replicate libraries and pool them after purification.

Q2: Can the kit be used for small RNA libraries?

• No. Small RNAs of about 22 nt are too short to be captured by the beads used in this workflow.

Q3: Is the kit compatible with FFPE samples?

• Yes. mRNA in FFPE samples may be degraded, so use rRNA depletion rather than poly(A) capture for RNA enrichment.

Q4: How many PCR cycles should I use?

• Follow the recommended cycle table (9–18 cycles depending on input and library type). If unsure, measure 1 μl of unpurified amplification product on a fluorometer and add cycles based on the actual yield.

Q5: Why does my library trace show a double peak?

• Possible causes are RNA degradation leading to nonspecific amplification; uneven fragment distribution after fragmentation of RNA from some species; or assessing the library on a high-sensitivity chip. Use an Agilent DNA 1000 kit, or dilute the library appropriately before running a high-sensitivity DNA kit.

Q6: What does a peak at 90–140 bp mean?

• It indicates adapter-dimer contamination. Dilute the library to 50 μl with nuclease-free water and repeat the 0.9× bead purification.