Truseq rna ht sample prep kit

RNA was extracted from various tissue types (5 floral developmental stages, mature flower, leaf, root and capsule) using TaKaRa kit for RNA extraction. The cDNA libraries were constructed from RNA. The mRNAs were filtered from total RNA using Oligotex mRNA Midi Kit (QIAGEN, GERMANY). RNA was quantified on Nano-Drop 2000 spectrophotometer (Thermo Scientific, USA) and the cDNA libraries were generated following Illumina manufacturing protocol as suggested previously [62 ]. In short, mRNAs were obtained from total RNA and fragments were made to an approximate length of 200 bp. Then, the isolated mRNAs were subjected to cDNA synthesis of first and second strand followed by adapter ligation and low-cycle enrichment following the instructions by TruSeq®RNA HT Sample Prep Kit (Illumina, USA). The library products thus purified were evaluated using the Agilent 2200 TapeStation and Qubit®2.0 (Life Technologies, USA), followed by dilution to 10 pM for in situ cluster generation on the HiSeq2500 pair-end flow cell and then pair end sequencing (2 × 100 bp) was performed. About 60 million reads were produced for each sample. Transcriptomic de novo assembly was performed using Trinity program with default parameters [63 (link)].

RNA was extracted from 9 tissues (5 FD stages, flower, silique, root, and leaf) using a TaKaRa RNA extraction kit, followed by the construction of cDNA libraries. Oligotex mRNA Midi Kit (QIAGEN, Hilden, Germany) was used to filter mRNAs from total RNA. RNA quantity and quality was checked on a Nano-Dropt 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), followed by cDNA library preparation using Illumina manufacturing protocol. The mRNAs were fragmented to an approximate length of 200 bp. The first and second strand cDNA was synthesized from the isolated mRNAs, followed by adapter ligation and low-cycle enrichment according to TruSeq^®RNA HT Sample Prep Kit (Illumina, San Diego, CA, USA). The purified library products were evaluated using Agilent 2200 TapeStation and Qubit^®2.0 (Life Technologies, Waltham, MA, USA) and diluted to 10 pM to generate clusters in situ on the HiSeq2500 pair-end flow cell. Sequencing (2 × 100 bp) was performed. Each sample produced about 60 million reads. Transcriptome de novo was done using the Trinity program with default parameters [29 (link)].

The primary aim of this study was to identify the genes responsible for low-temperature-induced flowering. The cDNA libraries were prepared from RNAs isolated from floral buds before and after 40-day cold treatment. The mRNAs were purified from total RNA using the Oligotex mRNA Midi Kit (QIAGEN, Germany) and quantified using a Nano-Drop 2000 spectrophotometer (Thermo Scientific, USA) to generate the cDNA library according to the Illumina manufacturer’s instructions as in our previous work [28 ]. Briefly, mRNAs were isolated from Total RNA and fragmented to approximately 200 bp. Subsequently, the collected mRNAs were subjected to first strand and second strand cDNA synthesis following by adaptor ligation and enrichment with a low-cycle according to instructions of TruSeq® RNA HT Sample Prep Kit (Illumina, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit®2.0(Life Technologies,USA) and then diluted to 10 pM for cluster generation in situ on the HiSeq2500 pair-end flow cell followed by sequencing (2 × 100 bp). An average of more than 60 million reads was generated for each sample.

For transcriptome sequencing, we constructed independent cDNA libraries for sepal, petal, labellum, and gynostemium obtained from floral buds at developmental stage 3 of three individual plants, separately. The floral apex resembles an inverted triangle, and the ventral outer sepal and petals grow quickly in this stage. Two replications were included in each sample to create eight multiplexed cDNA libraries. The mRNAs were purified from total RNA using Oligotex mRNA Midi Kit (QIAGEN, Germany) and quantified using Nano-Drop 2000 spectrophotometer (Thermo Scientific, United States) to generate the cDNA library according to Illumina manufacturer’s instructions (Yang and Zhu, 2015 (link)). The purified mRNAs were fragmented to approximately 200 bp and subjected to first strand and second strand cDNA synthesis, followed by adaptor ligation and low-cycle enrichment using TruSeq^® RNA HT Sample Prep Kit (Illumina, United States). The purified library products were evaluated with Agilent 2200 TapeStation and Qubit^®2.0 (Life Technologies, United States) and diluted to 10 pM for cluster generation in situ on the HiSeq3000 pair-end flow cell, followed by sequencing (2 × 100 bp). An average of more than 75 million reads were generated for each sample.