Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana

ABSTRACT: We sequenced the total RNA from a tissues mixed sample (inflorescences, rosette leaves, cauline leaves and stems) of Arabidopsis thaliana. After total RNA extraction, the same amount of tissue RNA were mixed. Ribosomal RNAs were deleted from the mixed tissue total RNAs using RiboMinus™ Plant Kit repeated three times. We also sequenced 9 poly(A)- RNAs from seedlings treated with different stress conditions at different times. The poly(A)- RNAs were collected by removing poly(A)+ RNAs four times . Then rRNAs were removed from poly(A)- RNAs three times. Sequencing of total RNAs from the mixed tissues sample is taken as an example of collecting novel transcripts from high-throughput data, which could be clarified by noncoding scores derived from our integrative models. Sequencing of nonpolyA RNAs are used for identifing stress-responsive nonpolyA lncRNAs and for validation of predicted lncRNAs.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Zhi Lu

PROVIDER: E-GEOD-49325 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features.

Di Chao C Yuan Jiapei J Wu Yue Y Li Jingrui J Lin Huixin H Hu Long L Zhang Ting T Qi Yijun Y Gerstein Mark B MB Guo Yan Y Lu Zhi John ZJ

The Plant journal : for cell and molecular biology 20141021 5

Recently, in addition to poly(A)+ long non-coding RNAs (lncRNAs), many lncRNAs without poly(A) tails, have been characterized in mammals. However, the non-polyA lncRNAs and their conserved motifs, especially those associated with environmental stresses, have not been fully investigated in plant genomes. We performed poly(A)- RNA-seq for seedlings of Arabidopsis thaliana under four stress conditions, and predicted lncRNA transcripts. We classified the lncRNAs into three confidence levels accordin ...[more]

PMID: 25256571

Similar Datasets

Project description:To identify mRNAs associated with human PUM1 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM1. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with anti PUM1 specific antibody Bethyl Laboratories, #300-201A coupled to protein G (PUM1) sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibody (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used. For PUM1 RIPs, we performed three biological replicates with technical (dye swap) replicates (total six arrays).(~40 ug aaRNA from PUM1 RIPs, ~9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays.Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel.To identify transcripts that were specifically enriched by association with PUM1, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 20 ug goat anti-PUMILIO 1 Bethyl Laboratories, #300-201A Keywords: replicate_design Computed

Project description:To identify mRNAs associated with human PUM2 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM2. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with specific anti PUM2 antibody coupled to protein A sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibodies (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used.For PUM2 RIPs, we performed four biological replicates but omitted the dye swaps due to the lower aaRNA obtained after amplification (~10 ug aaRNA from PUM2 RIPs, 9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays. Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel. To identify transcripts that were specifically enriched by association with PUM2, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 50 ug rabbit anti-PUMILIO 2 (Bethyl Laboratories, #300-202A) Keywords: replicate_design Computed

Dataset Information

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana

Publications

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets