title: GSM8146034: variegated edge leaves, replicate 2 [VE2]; Yucca flaccida; RNA-Seq
library_construction_protocol: Total RNA was extracted using the NucleoSpin RNA Plus kit according to the manufacturer's instructions (Macherey-Nagel, Duren, Germany), with minus modifications (2% PVP40 in lysis buffer) (en attente quantité ARNm pour libraiire - quesiton posée à novogene) After QC procedures, mRNA is enriched using oligo(dT) beads. The mRNA is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamer and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H, and Escherichia coli polymerase I to generate strand by nick-translation. The final cDNA libnrary is ready after a round of purification, terminal-repair, A-tailing, ligation of sequenciing adpaters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorescent (Life technologies), then diluted to 1ng/µl before checking insert size on an Agilent 2100 and quantifying to greater accurancy by quantitative PCR(Q-PCR).