title: GSM3104420: Circumbracteal nectary presecretory control replicate3 (1); Gossypium hirsutum; RNA-Seq
library_construction_protocol: RNA isolation: For a single biological replicate, approximately five nectaries were transferred with a clean forcep to a 2 ml Lysing matrix A tube (MP Biomedicals, Ref # 6910-500) containing a ceramic bead, resting in a liquid nitrogen bath. The tubes were quickly transferred to a QuickPrep adaptor (containing dry ice) and attached to the FastPrep 24™-5G  (MP Biomedicals) benchtop homogenizer. The tissue were subjected to 5 to 6 homogenizing runs of 40 seconds each at 6 m/sec, with each run interjected with a period of immersing the tubes in liquid nitrogen and refilling the adaptor with dry ice. Post-pulverization, 600µl of the RNA lysis buffer of the Quick-RNA™ MiniPrep kit (Zymo Research, Cat# R1054) was quickly added to the Lysing matrix tube and the tubes were vortexed. This was followed by addition of 50µl of the Plant RNA Isolation Aid (Thermo Fisher Scientific Cat#AM9690; erstwhile Ambion) and a quick vortex to aid removal of common plant contaminants such as polyphenolics and polysaccharides. Quick-RNA™ MiniPrep kit directions were followed for RNA isolation. Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality for all samples prior to submission to the University of Minnesota Genomics Center for barcoded library creation and Illumina HiSeq 2500 sequencing. All libraries were pooled and sequenced across two and a half lanes, then sequencing was repeated for quality concerns. This generated ≥24 M reads for each sample and the average quality scores were above Q30. Illumina's Truseq RNA Preparation v2 kit (RS-122-2001)