Product Description
The VAHTSTM Total RNA-seq (H / M / R) Library Prep Kit for Illumina® is a special strand-specific transcriptome library construction kit for the Illumina high-throughput sequencing platform. This kit is suitable for the amount of starting templates. The total RNA of humans, mice and rats is 0.1 to 1 µg. The difference from the conventional transcriptome library is that this kit can combine rRNA (including cytoplasmic rRNA 28S, 18S, 5S and mitochondrial rRNA 16S, 12S, 5.8S). ) It is removed from total RNA, leaving mRNA and other non-coding RNAs.
It is used for the analysis of non-coding RNA, such as LncRNA; Degraded RNA samples can also be used for library construction with this kit. When the second-strand cDNA is synthesized in this kit, dUTP is added to label it and the second tagged strand is digested with the UDG enzyme prior to PCR, so the sequencing information comes from the first-strand cDNA, resulting which ensures the directionality of the strand. RNA Analysis Analyze the sequencing data to determine whether the transcript is from the sense or antisense DNA strand.
The kit includes three independent packages of NR4, NR5 and NR6, which contain the library construction process. All necessary enzymes, buffers, and components have undergone strict quality control and functional verification, ensuring the stability and repeatability of the library construction to the greatest extent possible. The DNA and RNA purification magnetic beads and adapters must be prepared separately.
Adapters can be selected from VAHTSTM RNA Adapters (Vazyme # N803, Vazyme # N804 or Vazyme # N809, Vazyme # N810, Vazyme # N811, Vazyme # N812). For details on the adapter selection, see Common problems and solutions 6 (Page 17); Magnetic DNA Purification Beads can choose from VAHTSTM DNA Clean Beads (Vazyme # N411) or Agencourt® AMPure® XP Reagent (Beckman # A63880, # A63881, # A63882); RNA Purification Magnetic Beads can be chosen from VAHTSTM RNA Clean Beads (Vazyme # N412) or Agencourt® RNAClean® XP Beads (Beckman # A63987).
Fast: minimalist operating process reduces database creation time by 2 h
Compatibility: compatible with a variety of RNA enrichment modules, with any combination
General: flexible and free choice of chain-specific or non-chain-specific libraries
Storage: All components are stored at -20 ℃
mRNA purification and fragmentation
1. Bring mRNA Capture Beads to room temperature.
2. Prepare RNA sample by dissolving 0.1-1 μg total RNA in 50 μl nuclease-free water in a nuclease-free centrifuge tube, keeping on ice.
3. Mix the mRNA capture beads by inverting or vortexing, add 50 μl of the beads to the tube of total RNA and mix by pipetting 6 times.
4. Incubate the sample in a thermostatic device (i.e. PCR machine) at 65℃ for 5 minutes and then keep it at 4℃ to denature the RNA.
5. Allow the tube to stand at room temperature for 5 minutes, allowing the mRNA to bind to catch pearls.
6. Transfer the sample to a magnetic frame for 5 minutes and then carefully remove the supernatant without disturbing the capture beads.
7. Remove the sample from the magnetic frame, add 200 μl of Beads Wash Buffer and mix by pipetting 6 times. Return sample to magnetic frame, let stand for 5 min and carefully remove the supernatant without disturbing the capture beads.
8. Remove the sample from the magnetic frame, add 50 μl of Tris Buffer and mix thoroughly pipetting 6 times.
9. Incubate the sample in a thermostatic device (i.e. PCR machine) at 80℃ for 2 minutes and then keep it at 25℃.
10. Add 50 μl Beads, Binding Buffer, mix by pipetting 6 times.
11. Incubate at room temperature for 5 minutes.
12. Return the sample to a magnetic frame, let stand for 5 minutes and carefully remove the supernatant without disturbing the capture beads.
13. Remove the sample from the magnetic frame, add 200 μl of Beads Wash Buffer, and mix by pipetting 6 times. Let stand for 5 minutes on a magnetic frame and then Carefully remove the supernatant without disturbing the capture beads. Remove as much liquid as possible using a 10 µl pipette.
14. Remove sample from the magnetic frame, add 19.5 µL Frag/Prime Buffer, mix pipetting 6 times. Incubate the sample in a PCR device and set the programs according to the required chunk size:
- 150-200 bp insert: 8 minutes at 94℃, hold at 4℃.
- 200-300 bp insert: 5 minutes at 94℃, hold at 4℃.
- 250-450 bp insert: 6 minutes at 85℃, hold 4℃.
- 450-550 bp insert: 5 minutes at 85℃, hold 4℃.