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| Status |
Public on Apr 12, 2023 |
| Title |
diaphragm, mechanical ventilation without PEEP application, rep3 |
| Sample type |
SRA |
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| Source name |
diaphragm
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| Organism |
Oryctolagus cuniculus |
| Characteristics |
tissue: diaphragm
treatment: mechanical ventilation
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
PEEP0_3
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| Data processing |
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/, version:cutadapt-1.9).
We aligned reads of all samples to the oryctolagus cuniculus(ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/003/625/GCF_000003625.3_OryCun2.0) reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/, version:hisat2-2.0.4) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. HISAT2 allows multiple alignments per read (up to 20 by default) and a maximum of two mismatch when mapping the reads to the reference. HISAT2 build a database of potential splice junctions and confirms these by comparing the previously unmapped reads against the database of putative junctions.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/, version:stringtie-1.3.4d) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software (http://ccb.jhu.edu/software/stringtie/gffcompare.shtml, version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Assembly: OryCun2.0
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
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| Submission date |
Apr 02, 2023 |
| Last update date |
Apr 12, 2023 |
| Contact name |
Tongjian Zhao |
| Organization name |
School of Pharmaceutical Sciences, Jilin University
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| Street address |
1266 Fujin Road
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| City |
Changchun |
| ZIP/Postal code |
130021 |
| Country |
China |
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| Platform ID |
GPL25392 |
| Series (1) |
| GSE228739 |
Effect of mechanical ventilation and mechanical ventilationon with positive end expiratory pressure(PEEP) application on diaphragmatic dysfunction in rabbits |
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| Relations |
| BioSample |
SAMN34041305 |
| SRA |
SRX19844955 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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