E-MTAB-3736 - Membrane association and activity of rough endoplasmic reticulum associated RISC requires farnesylated chaperones

Status
Submitted on 5 March 2015, last updated on 13 July 2015, released on 31 July 2016
Organism
Arabidopsis thaliana
Samples (16)
Protocols (4)
Description
Aim and experimental set-up: Sequencing of small RNA from total RNA fractions. The aim of this experiment was to compare profiles of miRNA expression between the following genetic backgrounds: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter). Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested, and total RNA was prepared by Trizol extraction. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform. Sequencing of small RNA bound to AGO1 in membrane fractions The aim of this experiment was to characterize AGO1-bound small RNAs in membrane fractions, and to answer two specific questions: Can miRNAs be identified whose association with membrane-bound AGO1 is different between the following four genotypes: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter) Is the ratio between reads matching miRNA and miRNA* different between the above four genotypes? Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested. Microsomes were prepared from 2g of seedling tissue, solubilized by 1% deoxycholate and AGO1 was immunoprecipitated with specific antibodies (Agrisera). Immunopurified AGO1 was eluted from Protein A sepharose beads by competitive elution with antigenic AGO1 peptide. Small RNA was extracted by Trizol extraction from eluted AGO1. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform.
Experiment types
microRNA profiling by high-throughput sequencing, genetic modification design, replicate design
Contacts
Citation
Membrane association and activity of rough endoplasmic reticulum associated RISC requires farnesylated chaperones. Andrea Barghetti, Peter Brodersen.
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-MTAB-3736.idf.txt
Sample and data relationshipE-MTAB-3736.sdrf.txt
Links