Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq in Arabidopsis thaliana j3-1, j3-3, j2-2, and hcr3

ABSTRACT: RNA-prep of j3-1, j3-3, j2-2, hcr3 mutant from seedling or bud sample. RNA was extracted using the RNeasy plant (Qiagen, 74904) and sent to BGI Hongkong for RNA-seq library construction and sequencing. Briefly, mRNA enriched by using oligo dT beads were fragmented and used for cDNA synthesis. dUTP method was used for cDNA synthesis to make the RNA-seq library strand-specific. The constructed library was amplified to make DNA nanoball (DNB) and sequenced on DNBSEQ platform.

INSTRUMENT(S): DNBSEQ-G400

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Kyuha Choi

PROVIDER: E-MTAB-13417 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project 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.

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RNA-seq in Arabidopsis thaliana j3-1, j3-3, j2-2, and hcr3

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