2.6 Total RNA extraction and library preparation for wheat anthers of T. laevis and T. controversa
Anthers were collected from 5 different growth periods (0~150 μm, 150~400 μm, 400~700 μm, 700~1100 μm and 1100~1300 μm) frozen in liquid nitrogen and stored at -80 ˚C for later use. RN53-EASYspin Plus Complex Plant RNA Kit (Adlai, Beijing-China) was used to extract total RNA according to the manufacture’s instruction. The quality of the purified RNA was measured on the 260/280 nm and 260/230 nm absorbance ratio obtained, using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis was used to detect RNA integrity. RNA-seq transcriptome library was prepared by following TrueSeq RNA sample preparation kit from Illumina (San, Deigo, CA) using 1 µg of total RNA. The first cDNA synthesized using random primer (oligonucleotides) (Illumina, San Diego, CA, USA). Second-cDNA strand synthesized by DNA polymerase I, RNase H, dNTPs and buffer solution. cDNA fragments were purified using QIAquick PCR kit (Qiagen, Venlo, Netherland). These cDNA fragments washed by EB buffer for addition of end reparation Poly (A) and then ligated with special Illumina sequencing adapters. The final cDNA library was constructed by purification of the cDNA small fragments, which were enriched by PCR products. Sequencing was done by using Illumina HiSeq 2500 platform (Illumina, USA) by Shanghai Meiji Biomedical Technology Co., Ltd.