P-MEXP-1060 - P-MEXP-1060

Plant tissue was ground to fine powder using liquid nitrogen in a 2mL eppendorf tube. The sample was homogenized in 1 mL TRIzol Reagent by vortexing and incubated at room temperature for 5 min. Chloroform (200ul/1mL TRIzol) was added and the capped tubes were hand-shaken for 15 sec. They were incubated for 2-3 min, then centrifuged for 15 min at 11000rpm at 4oC. The aqueous phase was transferred to a new 1.5 mL tube and 500ul of isopropanol was added. The samples were incubated for 10 min at room temperature then centrifuged for 10 min at 11000 rpm at 4oC. The supernatent was removed and the pellet was washed with 1 mL of 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 min at 11000 rpm at 4oC. The pellet was air-dried for 10 min at room-temperature. The pellet was resuspended in 40 ul DEPC-treated water, incubated at 65oC for 10 min, and left on ice overnight at 4oC. They were centrifuged at 11000 rpm for 10 min and the supernatent was transferred to a new tube. Quality assessment was performed by spectrophotometer at OD 260 and OD 280. 5 ug of total RNA was amplified to aRNA using the Ambion MessageAmp Kit (ambion, #1750).
Amplification, Extracted product
All experiments using protocol P-MEXP-1060: (E-CAGE-2, E-CAGE-3, E-CAGE-4)