Salt-responsive miRNAs and target genes of Salix matsudana Koidz
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ABSTRACT: In order to screening the responsive miRNAs and target genes of willow under salt stress, the 30-day-old plants were exposed to the salt solution (100 mmol L-1 NaCl) for 0 h and 2 d. then RNA isolated from root and stem tissues for the same time point were mixed respectively in equal amounts for small RNA (sRNA) sequencing. sRNAs with 16–30 nt were separated from 1 µg total RNA by size fractionation. Subsequently, the selected sRNA fragments were ligated with specialized adaptors to the 5’ and 3’ ends (Illumina) using T4 RNA ligase. Then, the ligated RNAs were reverse transcribed and amplified for sequencing using Illumina Hiseq2500 (LC Sciences, Hangzhou, China). Salt stress-responsive miRNAs were identified by comparing the expression levels of miRNAs between the two libraries. Equal amounts of all 2 RNA samples were mixed together to construct one degradome library, and then sent to Hangzhou LC-Bio Co., Ltd (Hangzhou, China) for sequencing by Illumina Genome Analyzer GA-I (Illumina, San Diego, CA, USA).
Project description:Construction of transcriptome sequencing library and transcriptome sequencing was completed by LC-Bio Technology Co., Ltd. (Hangzhou, China). The expression profile of Machado-joseph deubiquitinases (MJDs) family in heart tissues of Ang II mice.
Project description:Construction of transcriptome sequencing library and transcriptome sequencing was completed by LC-Bio Technology Co., Ltd. (Hangzhou, China). The expression profile of Machado-joseph deubiquitinases (MJDs) family in heart tissues of Ang II mice.
Project description:To evaluate the change of miRNA expression profile in DF-1 cells in respond to Infectious bursal disease virus (IBDV) infection, Deep sequencing was performed by LC Sciences (Hangzhou, China) on DF-1 cells infected with mock or IBDV Lx strain at an MOI of 1 for 24 h.
Project description:An TMT-based quantitative crotonylome analysis was performed on the crotonylated proteins enriched from the chloroplast extracts of the normal and salt-treated wheat seedling leaves by using affinity purification and LC-MS/MS. The crotonylated peptides from wheat chloroplast extracts were enriched by using anti-crotonyllysine mouse mAb (Clone 4D5) antibody (PTM BioLabs, HangZhou, China PTM-501 and Nano LC-MS/MS was performed by using a Dionex rapid-separation liquid chromatography system interfaced with a Q Exactive HF (Thermo Fisher Scientific).
Project description:Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution. The aim of this study is to investigate the small RNA transcriptome in Cunninghamia lanceolata. Total RNAs of seeds and calli were extracted using RNAiso-mate for plant tissue and RNAiso plus (Takara, Dalian, Liaoning, China), whereas total RNAs of seedlings, adult leaves and stems were isolated with the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), and were then treated with RNase-free DNase I (Promega, Madison, WI, USA). Equal amount of total RNAs from the 5 different samples were mixed to form a single RNA pool. Twenty micrograms of total RNAs from the pool were used and 16 to 30-nt sRNAs were purified using Novex 15% TBE-¬Urea gel (Invitrogen). Two adaptors were sequentially ligated to the 5' and 3' ends of purified sRNAs. The ligation products were further purified from Novex 10% TBE-Urea gel. Reverse transcriptase SuperScript II (Invitrogen) and high-fidelity DNA polymerase Phusion (New England Biolabs, Ipswich, MA, USA) were used in the following RT-PCR reaction. The amplification products were cut from Novex 6% TBE-Urea gel. The purified DNA fragments were used for sequencing on an Illumina 1G Genome Analyzer at the Beijing Genomics Institute, Shenzhen, China.
Project description:Whole RNA-sequencing analysis was performed and analyzed by Lc. Biotech Co. Ltd. (Hangzhou, China). Primary mouse microglia from the Con + circCon group, Con + circDYM group, LPS + circCon group, and LPS + circDYM group were collected in TRIzol. Total RNAs were extracted from Trizol. UMI technology was used to label each sequence fragment with sequence tags, which minimized the interference of duplication caused by PCR amplification on the quantitative accuracy of the transcriptome. RNA sequencing reads were aligned to the mouse genome (GRCh37/hg19) using the software Hisat2 (2.0.4). Transcript abundance was evaluated by calculating fragments per kilo base of exon per million fragments mapped (FPKM).
Project description:Whole RNA-sequencing analysis was performed and analyzed by Lc. Biotech Co. Ltd. (Hangzhou, China). hippocampus tissue from the PBS_WT group, PBS_Rag1-/- group, Ctr CD8+TSCM_Rag1-/- group, and CUS CD8+TSCM_Rag1-/- group were collected in TRIzol. Total RNAs were extracted from Trizol. UMI technology was used to label each sequence fragment with sequence tags, which minimized the interference of duplication caused by PCR amplification on the quantitative accuracy of the transcriptome. RNA sequencing reads were aligned to the mouse genome (GRCh37/hg19) using the software Hisat2 (2.0.4). Transcript abundance was evaluated by calculating fragments per kilo base of exon per million fragments mapped (FPKM).
Project description:Whole RNA-sequencing analysis was performed and analyzed by Lc. Biotech Co. Ltd. (Hangzhou, China). hippocampus tissue from the Ctrl + Ctrl group, Ctrl + circDYM group, CUS + Ctrl group, and CUS + circDYM group were collected in TRIzol. Total RNAs were extracted from Trizol. UMI technology was used to label each sequence fragment with sequence tags, which minimized the interference of duplication caused by PCR amplification on the quantitative accuracy of the transcriptome. RNA sequencing reads were aligned to the mouse genome (GRCh37/hg19) using the software Hisat2 (2.0.4). Transcript abundance was evaluated by calculating fragments per kilo base of exon per million fragments mapped (FPKM).
Project description:In this study, C. gigantea miRNAs and their target genes were investigated by extracting RNA from young roots, tender stems, young leaves, and flower buds of C. gigantea to establish a small RNA (sRNA) library and a degradome library to further sequence. This study identified 194 known miRNAs belonging to 52 miRNA families and 23 novel miRNAs. Among the miRNA families, 158 miRNAs from 27 miRNA families were highly conserved and existed in a plurality of plants. In addition, 60 different targets for 30 known families and one target for novel miRNA were identified by high-throughput sequencing and degradome analysis in C. gigantea. Our analyses showed that conserved miRNAs have higher expression levels and more family members as well as more targets than other miRNAs. Meanwhile, these conserved miRNAs were found to be involved in auxin signal transduction, regulation of transcription, and other developmental processes in plants, which will help further understanding regulatory mechanisms of C. gigantea miRNAs. The samples were collected from the young roots, tender shoots, young leaves and flower buds of wild C. gigantea growing in Jiangsu Province. TRIzol reagent (Invitrogen, USA) was used to extract the total RNAs [20]. An Illumina next-generation sequencing system, i.e. the 1 G Genome Analyzer sequencing platform, was utilized for sRNA sequencing. An Illumina HiSeq 2000 (LC Sciences, USA) was used for degradome sequencing.
Project description:Unilateral femoral artery occlusion (right side) and a sham operation on the contralateral (left) side was performed in C57BL/6J mice under anesthesia by double ligation of the superficial femoral artery proximal to the deep femoral artery and distal femoral artery. Animal numbers are stated with the different experimental results. Total RNA was isolated from the distal adductor muscles by phenol-chloroform isolation (TRIzol, Invitrogen, Carlsbad, CA) at baseline and at 5 time points after femoral artery ligation (6h, 24h, 3 days, 7 days, 14 days) from 5 mice per time point. RNA was pooled in equal amounts, and microarray analysis for all identified murine miRNAs (miRBase 9.0) was performed by a service provider (LC Sciences, Houston, TX).