title: ncRNA_Seq of Batrachium bungei: young fresh leaf
design: Total RNA was extracted from the tissue using TRIzol Reagent (Plant RNA Purification Reagent for plant tissue) according the manufacturers instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). RNA degradation and contamination was monitored on 1%agarose gels. Then RNA quality was determined by 2100 Bioanalyser (Agilent Technologies) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample (OD260/280=1.8~2.2, OD260/2302.0, RIN8.0, 28S:18S1.0, >1g) was used to construct sequencing library. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturers instructions (Illumina, San Diego, CA). RNA-seq transcriptome strand library was prepared following TruSeqTM Stranded Total RNA Library Prep Kit from Illumina (San Diego, CA) using 1g of total RNA. Shortly, ribosomal RNA (rRNA) depletion instead of poly(A) purification was performed by Ribo-off rRNA Depletion Kit and then fragmented by fragmentation buffer firstly. Secondly first-stranded cDNA was synthesized with random hexamer primers. Then we removed the RNA template and synthesized a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. The incorporation of dUTP quenched the second strand during amplification, because the polymerase did not incorporate past this nucleotide. AMPure XP beads were used to separate the ds cDNA from the second strand reaction mix. A single A nucleotide was added to the 3 ends of these blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. Lastly, multiple indexing adapters were ligated to the ends of the ds cDNA. Libraries were size selected for cDNA target fragments of 200300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15PCR cycles. After quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina Novaseq 6000 sequencer (2150 bp read length).