biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsFloyd Wittinktranscription profiling by arrayArabidopsis thalianaArabidopsis thalianahttps://www.ebi.ac.uk/biostudies/studies/E-CAGE-42Arabidopsis thaliana cv Ler is grown on agar,seedlings are sampled during development according to Paradigm index (stage 0.7-1.02-1.04-1.08) as part of CAGEbiostudies-arrayexpressGrowth Protocol - Arabidopsis thaliana seeds were incubated on agar containing 0.5 x MS minerals and vitamins; 0.1% sucrose; 0.5 g/l MES, pH 6.0;6 g/l Lab M agar for 3 days at 4 C. On the third day, plates were removed from the cold room and incubated in groth chambers conditioned at 20 C, 65 % humidity with cool while light at 100 uEm-2s-1, and a 16 hour/ 8 hour day/night photoperiod. Samples were collected when a phenotype as defined by Paradigm index was reached. Samples were immmediately frozen in liquid nitrogenNucleic Acid Extraction - Plant issue was ground to fine power using liquid nitrogen in a 2ml eppendorf tube. Samples were homogenized in TrIzol Reagent (invitrogen): 1 ml for 100 mg of plant tissue, by vortexing and incubated at room temperature for 5 minutes. Chloroform (200 ul/ ml TRIzol) was added and the capped tubes were vortexed for 5 seconds. Samples were incubated for 2-3 minutes, centrifuged for 15 minutes at 11000 rpm at 4 C. The aquous phase was transferred to a new 1.5 ml tube and 500 ul isopropanol was added. Samples were incubated for 10 minutes at room temperature before centrifugation at 4 C for 10 minutes at 11000 rpm. The supernatant was removed and the pellet was washed with 1 ml 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 minutes at 11000 rpmLabeling - 5 ug of aRNA (11.5 ul) is mixed with 1 ul of random nonamers (GeneLink; 1ug/ul); 1 ul of test spikes (Lucidea Score Card, Amersham BioSciences)and 0.5 ul RNaseOUT (Invitrogen), denatured for 10 minutes at 70 C and chilled on ice. The following components were added to the smple: 5 ul of 5 x first strand buffer;1 ul of 10 mM GAT-TP/2 mM CTP; 2.5 ul 0.1 M DTT; 1.5 ul of cy5-dCTP (Amersham Pharmacia Biotech, 25 nmole); 2 ul Superscript II RT (200 U/ul, Invitrogen). This mixture was incubated for 2.5 hours at 42 C and chilled on ice. Samples were denatured by adding 2 ul of 2.5 M NaOH for exactly 15 minutes at 37 C, then 10 ul 2 M MOPS was added and the samples were put on ice. After the addition of 5 ul 3 M NaAc pH 5.0, the dyes were purified with Qiaquick Minelute PCR purificationNucleic Acid Extraction - forward extension primers as defined by CATMA were end-labeled with cy3Sample Processing - Arabidopsis thaliana seeds are grown until a developmental stage as defined by Boyes et al.Hybridization - CATMA mirror slides (Amersham Biosciences) were prehybridized in a solution of 1% BSA;0.1%SDS;5 x SSC) at 42 C for 1 hour. The slides were dipped 5 times in distilled water at room temperature, then in isopropanol and centrifuged for 3 minutes at 750 g to dry the slides. Slides were placed in a Corning CMT hybridization chamber with a 25 x 60 Lifterslip and 20 ul of distilled water in each groove to keep the chamber humid. The target was diluted to a final volume of 90 ul as follows: 20 ul of purified labeld cDNA, 20 ul 20x SSC, 45 ul formamide and 0.8 ul 10 %SDS. The target is denatred at 96 C for 3 minutes and directly cooled to 4 C. The target mixture was put on the chip as quick as possible. The microarray is sealed in a chamber and submerged in a 42 C waterbath for appLabeling - cage_oligo_reference was end-labeld with cy3 during oligo synthesisMIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsFloyd WittinkfalseTranscription profiling of Arabidopsis Ler seedlings sampled during developmentArabidopsis thaliana cv Ler is grown on agar,seedlings are sampled during development according to Paradigm index (stage 0.7-1.02-1.04-1.08) as part of CAGE2006-10-31T00:00:00Z2022-03-07T03:25:20.94Z2022-03-07T03:25:20.94ZE-CAGE-42EFO_0002768