Qubit 2.0 fluorometer rna assay

Intact ovaries were dissected from fixed florets on ice using fine tweezers to avoid physical damage. Ovules were subsequently isolated by pulling apart the bifurcated stigma to separate the ovary wall from the ovaries. Fifty ovules per sample were collected. Ovule RNA was extracted using RNeasy Plant Mini Kit (QIAGEN) following the manufacturer’s protocol. The quantity and quality of RNA were checked using the Qubit 2.0 Fluorometer RNA assay (Invitrogen) and an Agilent 2100 Bioanalyzer using a RNA 6000 nano kit (Agilent Technologies), respectively. For each sample, 500 ng of total RNA was used for poly-A capture, cDNA synthesis and amplification using KAPA stranded RNA-seq Kit. Libraries were quality checked with the Qubit 2.0 Fluorometer dsDNA high sensitivity assay (Invitrogen) and Fragment Analyzer Automated CE System (Agilent Technologies), and were sequenced on an Illumina NextSeq (300 Cycles) PE150 Mid Output flow cell on which three biological replicates of two genotypes were pooled.

RNA-seq was performed thrice, for decreasing [LB_0.75x, LB_0.5x, and LB_0.25x, at 180 min; LB_0.5x at 60 and 125 min] and for increasing (LB_1.5x, LB_2.0x and LB_2.5x, at 180 min) medium richness relative to a control (LB_1.0x) (an independent control was used for each three sets of conditions). Cells from 3 independent biological replicates of MG1655 in each modified medium were treated with RNA protect bacteria reagent (Qiagen, Germany), to prevent degradation of RNA, and their total RNA was extracted using RNeasy kit (Qiagen). RNA was treated twice with DNase (Turbo DNA-free kit, Ambion) and quantified using Qubit 2.0 Fluorometer RNA assay (Invitrogen, Carlsbad, CA, USA). Total RNA abundance was determined by gel electrophoresis, using a 1% agarose gel stained with SYBR safe (Invitrogen). RNA was detected using UV with a Chemidoc XRS imager (Biorad).