Purified mRNA isolated from the control sample and from the cold-treatment mixture were separately fragmented with divalent cations under increased temperature. These short fragments were taken as templates to synthesize the first-strand cDNA using hexamer primers and superscript™III (Invitrogen™, Carlsbad, CA, USA). Second-strand cDNA was then synthesized in a solution containing buffer, dNTP, RNaseH and DNA polymerase I and subsequently purified using a QiaQuick PCR extraction kit (Qiagen). EB buffer was used to resolve these short fragments for end reparation and poly (A) addition. The sequence adaptors were linked to two ends of short cDNA sequences and suitably sized cDNA fragments were selected out for PCR amplification based on the agrose gel electrophoresis results. Finally, the library established was sequenced using an Illumina Hiseq™ 2000. The paired-end library was developed according to the paired-End sample Preparation Kit protocol (Illumina, USA). The transcriptome short reads were de novo assembled software following the protocol documented by Grabherr et al. [18 ].
Hexamer primers and superscript 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hexamer primers and Superscript III are laboratory reagents used in reverse transcription and PCR (Polymerase Chain Reaction) applications. Hexamer primers are short DNA sequences that bind to RNA templates, initiating the synthesis of complementary DNA (cDNA) strands. Superscript III is a thermostable reverse transcriptase enzyme that catalyzes the conversion of RNA into cDNA, which can then be amplified and analyzed using PCR techniques.
Automatically generated - may contain errors
4 protocols using hexamer primers and superscript 3
1
RNA-seq Transcriptome Assembly Protocol
2
Illumina RNA-seq Library Preparation
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Purified mRNA was separately fragmented with divalent cations under increased temperature. These short fragments were taken as templates to synthesize the first-strand cDNA using hexamer primers and superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was then synthesized in a solution containing buffer, dNTP, RNaseH and DNA polymerase I and subsequently purified using a QiaQuick PCR extraction kit (Qiagen). EB buffer was used to resolve these short fragments for end reparation and poly (A) addition. The sequence adaptors were linked to two ends of short cDNA sequences and suitably sized cDNA fragments were selected out for PCR amplification based on the agrose gel electrophoresis results. Finally, the library established was sequenced using an Illumina Hiseq 200. The paired-end library was developed according to the paired-end sample Preparation kit protocol (Illumina, USA).
3
Illumina Paired-End RNA Sequencing
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The initial step involved the fragmentation of purified mRNA, achieved through the application of divalent cations at an elevated temperature. These resulting shorter fragments were employed as templates for the synthesis of the first-strand cDNA, utilizing hexamer primers and superscript III (Invitrogen, Carlsbad, CA, USA). Subsequently, the second-strand cDNA was synthesized in a solution containing buffer, dNTP, RNaseH, and DNA polymerase I. The synthesized cDNA was then purified using a QiaQuick PCR extraction kit (Qiagen, Hilden, Germany) with a buffer containing dNTP, RNaseH, and DNA polymerase I. To facilitate end reparation and poly(A) addition, EB buffer was utilized to resolve these short fragments. Sequence adaptors were ligated to both ends of the short cDNA sequences, and appropriately sized cDNA fragments were selected for PCR amplification based on the results of agarose gel electrophoresis. Finally, the established library was sequenced using an Illumina Hiseq 2000. The paired-end library was prepared following the protocol provided by the Paired-End Sample Preparation Kit (Illumina, San Diego, CA, USA).
4
Transcriptome Sequencing and Assembly
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Purified mRNA isolated from the control sample and from the cold-treatment mixture were separately fragmented with divalent cations under increased temperature. These short fragments were taken as templates to synthesize the first-strand cDNA using hexamer primers and superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was then synthesized in a solution containing buffer, dNTP, RNaseH and DNA polymerase I and subsequently purified using a QiaQuick PCR extraction kit (Qiagen). EB buffer was used to resolve these short fragments for end reparation and poly (A) addition. The sequence adaptors were linked to two ends of short cDNA sequences and suitably sized cDNA fragments were selected out for PCR amplification based on the agrose gel electrophoresis results. Finally, the library established was sequenced using an Illumina Hiseq 2000. The paired-end library was developed according to the Paired-End sample Preparation kit protocol (Illumina, USA). The transcriptome short reads were de novo assembled using transcriptome de novo assembly software following the protocol documented by Grabherr et al (2010) [29] , using default Kmer value (K = 25).
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