title: L20
organism: Triticum aestivum
description: Protocols: Having cut both side of embryo and another, the pericarp of the seed were carefully stripped and frozen in liquid nitrogen and stored at −80˚C until RNA extraction. Plants of purple-grain wheat line Luozhen No.1 were grown under natural field conditions at Shandong Agricultural University Experimental Station (36°169 N, 117°169 E), Tai'an, China during October, 2010 to June, 2011. The spikes of purple-grain wheat Luozhen No.1 were bagged with four-layer Kraft paper bags after pollination. Raw reads produced from sequencing machines contain dirty reads which contain adapters, unknown or low quality bases. Dirty raw reads are discarded and generated clean reads. Transcriptome de novo assembly is carried out with short reads assembling program - Trinity. Trinity firstly combines reads with certain length of overlap to form longer fragments, which are called contigs. Then the reads are mapped back to contigs; with paired-end reads it is able to detect contigs from the same transcript as well as the distances between these contigs. Finally, Trinity connects the contigs, and get sequences that cannot be extended on either end. Such sequences are defined as Unigenes. When multiple samples from a same species are sequenced, Unigenes from each sample's assembly can be taken into further process of sequence splicing and redundancy removing with sequence clustering software to acquire non-redundant Unigenes as long as possible. Total RNAs were extracted from pericarp of purple-grain wheat using an RNeasy Plant Mini Kit according to the standard protocol (Cat. No. 74904, Qiagen, Valencia, CA, USA). At least 500 ng total RNA was extracted from each material used for library construction. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeqTM 2000.