RNAseq of Euphorbia peplus: root biorep 3
Organs were flash frozen in liquid nitrogen immediately after harvesting, ground into a fine powder, and then processed for RNA extraction using a combined TRI Reagent (Sigma-Aldrich, Saint Louis, MO, U.S.A.) and Monarch Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, U.S.A.) protocol. One ml of TRI Reagent was added to approximately 50 mg of ground tissue, the samples were vortexed for 30 seconds, and then 200 l of Chloroform was added. The samples were vortexed 3 x 30 seconds, left to sit at room temperature (RT) for 5 minutes, centrifuged at 12,000 G for 15 minutes at 4 C, and then the aqueous phase (the top layer) was transferred into a one-to-one mix with 25 Phenol:24 Chloroform:1 Isoamyl Alcohol (77617, Sigma-Aldrich, Saint Louis, MO, U.S.A.). The samples were vortexed for 30 seconds, and then centrifuged at 21,000 G for 10 minutes at 4 C. The top layer was transferred directly onto gDNA removal columns provided by the NEB Monarch Total RNA Miniprep Kit, and manufacturer guidelines were followed for Part 2 (RNA binding and elution) of the Monarch prep kit. RNA quality and quantity were accessed using a DeNovix DS-11 FX+ spectrophotometer (DeNovix Inc., Wilmington, DE, U.S.A.). To construct RNA-seq libraries for Illumina sequencing, mRNA was isolated from 1,000 ng of total RNA using a NEBNext Poly(A) mRNA Isolation Module (E7490, New Englad Biolabs, Ipswich, MA, U.S.A.), followed directly by library construction using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420). The libraries were barcoded with NEBNext Multiplex Oligos for Illumina Set 1 #E7335. The libraries were submitted to the Cornell Institute for Biotechnology Genomics Center, where they were quantified using qRT-PCR, quality checked on a Bioanalyzer (Agilent, Santa Clara, CA, U.S.A.), and pooled in equimolar ratios for 12-plex sequencing on a NextSeq 500 (Illumina, Hayward, CA, U.S.A.) 2x150 paired-end run.