AccuNext Chain-Specific Single Cell/Micro RNA Construction Kit (Illumina)

This product is a strand-specific RNA sequencing library construction kit designed for illumina's high-throughput sequencing platform. It can be used to construct RNA libraries from 1-100 human cells or 10 pg ~ 10 ng of Human Total RNA samples, and it contains all the necessary components for RNA fragmentation, reverse transcription (RT), PCR library amplification and ribosomal RNA removal. RNA is fragmented by heating, then reverse transcribed using a 3′-N6 Primer with a universal junction sequence, which utilizes the terminal transferase activity of the reverse transcriptase enzyme to make a specific junction at the end of the cDNA; PCR amplification is performed using primers with illumina sequencing junctions [e.g.AccuNext CDI-joining primers (Illumina, for RNA libraries) (Code No. AG12505, AG12506, AG12507)], followed by ribosomal RNA removal, and then two rounds of amplification using universal primers, and then purification can be completed to obtain sequencing libraries for illumina sequencing platform.
Since rRNA accounts for nearly 90% of Total RNA, it is necessary to remove rRNA in order to obtain more effective data volume and reduce sequencing costs. The reaction system of this product has been optimized to digest DNA fragments derived from ribosomal rRNA by using rRNA Depletion Probes and rRNA Depletion Enzyme (Probes are designed based on human nuclear rRNA and mitochondrial rRNA), which is especially suitable for building libraries of cellular samples or trace RNA samples that need to remove rRNA. library construction.
This product is easy to operate, no need to carry out traditional end repair, junction connection and other cumbersome steps to obtain RNA sequencing libraries; it is suitable for library construction of low input and low quality RNA, no need to remove ribosomal RNA before library construction, the data obtained from sequencing is highly uniform and complete coverage; optimized reaction system improves the efficiency of library conversion; at the same time, the directionality of template conversion reaction retains the directionality of RNA strand, which can obtain RNA strand-specific sequencing data. At the same time, the orientation of the template conversion reaction preserves the orientation of the RNA strand, and RNA strand-specific sequencing data can be obtained.
The reaction system of this product has been carefully optimized, please use the reagents provided in this product for RNA fragmentation, reverse transcription, rRNA removal and PCR library amplification experiments, it is not recommended to change the dosage and concentration of any of the reaction components or to replace the components in this product with other equivalent products, in order to avoid obtaining bad results. If substitution is required, please verify first.

1. For illumina sequencing platform: integrating Index and illumina connectors, constructed RNA libraries can be directly sequenced by NGS.
2. High sensitivity: RNA library construction from single cells and 10 pg Human Total RNA.
3. Widely used: suitable for degraded samples, compatible with RNA samples with RIN value 2 ~ 10. Especially for micro samples or degraded RNA samples, which are unable to remove rRNA before library construction, this product can be used to complete RNA library construction.
4. Simple operation: RNA sequencing libraries can be obtained without the traditional tedious steps such as end repair and junction connection.
5. Accurately recognizes RNA strand specificity: Read 1 reads the antisense strand sequence of the original RNA, and Read 2 reads the positive-sense strand sequence of the original RNA.

Package 2-1 components are as follows (stored at -80°C):

individual parts making up a compound	AG12526 (12 rxns)	AG12527 (48 rxns)
5 ‘- Adapter Primer Mix	54 μl	216 μl
rRNA Depletion Probes*1	15 μl	60 μl
Control Total RNA (1 μg / μl)*2	5 μl	5 μl

*1: rRNA Depletion Probes Avoid repeated freezing and thawing. If multiple uses are required, it is recommended that they be stored in appropriate volumes.
*2: Control Total RNA is 293T Cell Total RNA.

Package 2-2 is grouped as follows (stored at -20°C):

individual parts making up a compound	AG12526 (12 rxns)	AG12527 (48 rxns)
10X Cell Lysis Buffer	228 μl	920 μl
RNase Inhibitor ( 40 U/μl )	18 μl	72 μl
5X First-Strand Buffer	48 μl	192 μl
3′-N6 Primer	12 μl	48 μl
AccuNext Reverse Transcriptase ( 100 U/μl )	24 μl	96 μl
2X AccuNext PCR Buffer	900 μl	900 μl X 4 pcs
AccuNext DNA Polymerase ( 1 U / μl )	36 μl	144 μl
AccuNext PCR Primer Mix	24 μl	96 μl
10X rRNA Depletion Reaction Solution	30 μl	120 μl
rRNA Depletion Enzyme	24 μl	96 μl
Nuclease free water	1 ml	1 ml X 3 pcs

Attention:AccuNext CDI Junction Primers (Illumina, for RNA libraries) (Code No. AG12505, AG12506, AG12507) are required for the experiment, but are not configured in this product and need to be purchased separately.AccuNext Information on CDI-joining primers (Illumina, for RNA libraries) is provided in Appendix B and C.

Storage temperature: Package 2-1 -80℃ Storage Package 2-2 -20℃ Storage
Transportation Temperature: Package 2-1 Dry Ice Transportation Package 2-2 -20℃ Ice Pack Transportation or Dry Ice Transportation