Divalent cation fragmentation buffer

Newly emerged leaves from wild type and rubescent mutants were collected and total RNA was isolated using the cetyltrimethylammonium bromide (CTAB) method (Wang et al., 2011 (link)). Two cDNA libraries each for the wild type and rubescent mutant were prepared for 100 bp paired-end RNA-Seq transcriptome. cDNA library construction and sequencing analysis were performed by Hangzhou Woosen Biotechnology Co. Ltd (Hangzhou, Zhejiang, China).
Poly (A) mRNA was purified with oligo (dT) beads from total RNA, and then the mRNA-enriched RNA was randomly segmented into 200–700 nt fragments in a divalent cation fragmentation buffer (Illumina, Hayward, CA) for 8 min at 94°C. These short fragments were used as templates to synthesize the first-strand cDNA using random hexamer primers and the second-strand cDNA was generated using RNaseH and DNA polymerase I. Those short cDNA fragments were purified with QiaQuick PCR extraction kit and washed with elution buffer (EB) for end repairing and tailing-A. Then those short fragments were ligated to sequencing adapters according to Illumina's protocol (San Diego, CA, USA) and further separated by agarose gel electrophoresis. Fragments of 300–500 bp were enriched by PCR amplification to create a cDNA library.

RNA Keeper-ICE tissue transfer buffer was bought from vazyme company (Nanjing, China). Quick RNA Isolation kit was bought from Biotech Corporation (Beijing, China). AMPure XP beads were bought from Beckman (CA, USA). HiScript® II Q RT SuperMix reagent was bought from vazyme company (Nanjing, China). TRIzol reagent was bought from TransGen Biotech (Beijing, China). Divalent cation fragmentation buffer was bought from Illumina (CA, USA). RNaseH and DNA polymerase I were bought from TaKaRa (Kyoto, Japan). Elution buffer was bought from GENEWIZ (Suzhou, China). Agarose gel electrophoresis buffer was obtained by dissolving 1 g of Agarose in 100 mL of Tris Acetate EDTA electrophoresis buffer. Tris Acetate EDTA electrophoresis buffer was bought from TSINGKE (Beijing, China). Agarose was bought from Sigma (Darmstadt, Germany).