title: Transcriptome Sequencing and De Novo Analysis for Ma bamboo
design: Total RNAs were extracted from these materials using the Norgen RNA Purification Kit (Canada). The quality and quantity of total RNA was analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and gel electrophoresis. Equal quantities of high-quality RNA from each material were pooled for cDNA synthesis.   cDNA library construction for Illumina sequencing  The cDNA library was constructed following the manufacturer’s instructions (Illumina). In briefly, the poly-(A) mRNA was isolated from the total RNA samples with Magnetic Oligo (dT) Beads. To avoid priming bias, the mRNA was fragmented by the RNA fragmentation kit (Ambion) before cDNA synthesis. The cleaved RNA fragments were transcribed into first-strand cDNA using reverse transcriptase (invitrogen) and random hexamer-primers, followed by second-strand cDNA synthesis using DNA polymerase I (New England Biolabs, NEB) and RNaseH (invitrogen). The double-stranded cDNA was end-repaired using T4 DNA polymerase (NEB), the Klenow fragment (NEB), and T4 polynucleotide kinase (NEB) followed by a single < A > base addition using Klenow 3’ to 5’ exo-polymerase (NEB), then ligated with an adapter or index adapter using T4 DNA ligase (NEB). Adaptor-ligated fragments were separated by size on an agarose gel, and then the desired range of cDNA fragments (200±25 bp) were excised and retrieved using a Gel Extraction Kit (Axygen). PCR was performed to selectively enrich and amplify the cDNA fragments using Phusion Master Mix (NEB), and then libraries were prepared from a 150–200 bp size-selected fraction following adapter ligation and agarose gel separation. After validation with an Eppendorf Mastercycler ep realplex Real-Time PCR System, the cDNA libraries were sequenced using a single end read protocol with 32 bp of data collected per run on the Illumina Genome Analyzer IIx sequencing platform. Data analysis and base calling were performed by the Illumina instrument software.
library_name: Ma Bamboo