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| Status |
Public on Jul 17, 2017 |
| Title |
ΔUPF1a-Rep1 |
| Sample type |
SRA |
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| Source name |
Tetrahymena thermophila whole cell
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| Organism |
Tetrahymena thermophila |
| Characteristics |
genetic background: B2086
genotype: Somatic UPF1a gene knockout
knockout gene accession number: TTHERM_00726300
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| Growth protocol |
The Tetrahymena B2086 wild-type (WT) strain (obtained from the Tetrahymena Stock Center at Cornell University) and mutant strains (Supplemental Table 1) were maintained in Super Proteose Peptone (SPP) medium (1% Proteose peptone, 0.2% Glucose, 0.1% Yeast extract, 0.003% Sequestrene) at 30°C on a rotary shaker at 135 rpm.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 5ml of vegetative Tetrahymena WT and mutant cells (density: 2.5 x 105 cells/mL) using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA)
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structurewere ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers andIndex (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
The processed data for this sample contains data for both GSM2417231 and GSM2417232. DEXSeq output for wildtype vs UPK1a KO is available on the series record.
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| Data processing |
Basecalls performed using CASAVA version 1.8
The adaptor sequences were trimmed, and low-quality reads (contianing over 10% of N, or the quality score of over 50% of base is lower than 5) were discarded to get clean reads for downstream analysis
RNA-seq reads were aligned to the Tetrahymena macronuclear genome assembly (version:June2014) using Tophat (v2.0.9) with the following parameters : tophat -o -p 4 --coverage-search --library-type fr-unstranded.
Reference guided transcript assembly performed using Cufflinks (v2.1.1) with the following paprameters: cufflinks bam_file -g T_thermophila_June2014.gtf -p 10 -o. The "T_thermophila_June2014.gtf" file was downloaded from the Tetrahymena Genome Database (http://ciliate.org/index.php/home/downloads)
The genome annotation files generated from previous step using wild type RNA-seq reads and ΔUPF1a RNA-seq reads were merged with reference genome annotation file by cufflinks (v2.1.1) with following parameters: cuffmerge Genome_annotation_list (a list containing the path to genome annotation files) -g T_thermophila_June2014.gtf -p 20–o Merged.gtf
The merged annotation file generated from previous step was compared with reference genome annotation file (T_thermophila_June2014.gtf) using cuffcompare algorithm (included in Cufflinks v2.1.1) with default settings to identify potential novel isoforms.
The fragments per kilobase of transcript per million mapped reads (FPKM) for each gene and transcript was determined using cuffdiff algorithm with default settings.
The exon usage was determined using DEXSeq (v1.12.1) . Briefly, prior to the DEXSeq analysis, the .bam files were indexed and sorted using samtools with the following parameters (1. samtools index; 2. samtools view -h). The genome annotation file (merged.gtf) was then modified using dexseq_prepare_annotation.py script, a phython script provided in DEXSeq package. The sequencing reads assigned to each exon in WT and ΔUPF1a samples were calculated using a phython script provided in DEXSeq package, dexseq_count.py, with the following parameter: -p yes. At last, the exon usage was calculated using DEXSeq algorithm in R (v3.1.1). The FDR value was set to 0.05.
Genome_build: Tetrahymena macronuclear genome assembly (version:June2014; downloaded from Tetrahymena Genome Database)
Supplementary_files_format_and_content: Tab-delimited Excel files include FPKM values for each sample; WTvsUPF1aKO_DEXSeq.csv contains quantification of exon usage.
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| Submission date |
Dec 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Wei Miao |
| E-mail(s) |
miaowei@ihb.ac.cn
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| Organization name |
Institute of hydrobiology, CAS
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| Street address |
donghu south road #7
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| City |
wuhan |
| State/province |
hubei |
| ZIP/Postal code |
430072 |
| Country |
China |
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| Platform ID |
GPL15706 |
| Series (1) |
| GSE90899 |
Nonsense-mediated mRNA decay in Tetrahymena is EJC independent and requires a protozoa-specific nuclease |
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| Relations |
| BioSample |
SAMN06111675 |
| SRA |
SRX2396516 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2417231_Upf1a.xlsx |
5.8 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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