TAG_Leaf
Illumina sequencing using 2101 bp Paired End sequencing platform run on a single lane of the Illumina HIseq 2000 (Genomics Core, UZ Leuven, Belgium) in accordance with the manufacturers protocol. Briefly, the poly (A) mRNA was purified from 25 g of total RNA samples using Oligotex mRNA mini prep kit (Qiagen, Germany). The mRNA was sheared into 200500 bp short fragments using divalent cations at 94C for 5 min before cDNA synthesis. These short fragments were further used as templates and first strand of cDNA was synthesized using Superscript II reverse transcriptase (Life Technologies, Inc.) and random hexamer primers. Afterwards, the second strand cDNAs was synthesized by DNA polymerase I along with the RNase H to limit the RNA. Further this synthesized cDNA (now double stranded) is subjected to end repair and phosphorylation was done by using T4 DNA polymerase and Klenow polymerase. Afterwards, 3adenylation of repaired cDNA was done using Klenow 3-5 exopolymerase and Illumina HiSeq Adaptors were ligated using T4 DNA ligase. The final products were further purified and selected for PCR amplification to construct cDNA library. Finally, Illumina HiSeq 2000 (Genomics Core, UZ Leuven, Belgium) was used for sequence the cDNA library. Further, samples were demultiplexed and Illumina HiSeq adapters were trimmed using CASAVA v1.8.2 software (Illumina, Inc., USA) to procure high quality sequenced library.