GSM1202544: H3; Brassica napus; RNA-Seq
Gynoecia were removed and immediately conserved in RNAfixer Stabilization Reagent (BioTeke Corporation) for one week, then transferred to liquid nitrogen and kept at -80°C. Total RNA was extracted using the Polysaccharide and Polyphenol Total RNA Isolation Kit (spin column; Bioteke Corporation). RNA libraries were prepared for sequencing as the protocols: mRNA is enriched by using the oligo(dT) magnetic beads from the total RNA. Adding the fragmentation buffer, the mRNA is interrupted to short fragments (about 200 bp), then the first strand cDNA is synthesized by random hexamer-primer using the mRNA fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I are added to sythsize the second strand. The double strand cDNA is purified with QiaQuick PCR extraction kit and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors are ligated to the fragments. The required fragments is purified by agrose gel electrophoresis and enriched by PCR amplification.