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Status |
Public on Jul 31, 2018 |
Title |
Stage 4 rep5 [ncRNA] |
Sample type |
SRA |
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Source name |
oviduct luminal fluid
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Organism |
Bos taurus |
Characteristics |
tissue: oviduct luminal fluid estrous cycle stage: pre-ovulatory stage, days 18-20
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Growth protocol |
EVs were isolated by ultracentrifugation (Théry et al., 2006; Almiñana et al., 2017). First, flushing samples were centrifuged at 300 g for 15 min, followed by 2,000 g for 15 min, and then at 12,000 g for 15 min to remove cells, blood, and cell debris and ultracentrifuged twice at 100,000 g for 90 min (BECKMAN L8-M; SW41T1 rotor) to pellet EVs. The pellets were resuspended in 50 µL of PBS and stored at -80ºC for further analysis. Oviductal flushings from 3 animals were pooled for each replicate.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was isolated from a total of 20 EVs samples (4-5 replicates, 4 stages) using miRNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. RNA quality and concentration were evaluated by Agilent 2100 Bioanalyzer Nano and small RNA assay (Agilent Technologies, Santa Clara, CA.) and NanoDrop (ThermoFisher). A total of 444 ng RNA was used for the preparation of each small RNA library using NEXTflex™ Small RNA-Seq Kit v3 (Bioo Scientific). Library preparation followed the manufacturer's instructions.
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Library strategy |
ncRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina HiSeq 2500 |
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Description |
S4_R5 Total RNA enriched for shorter molecules. miRNA_isoforms_counts.txt miRNA_isoforms_summarized_counts.txt Filtered_sequence_counttable_smallRNA_exo.txt Filtered_sequence_counttable_smallRNA_exo_annotation_outliers_removed.txt
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Data processing |
Clip adapter (Version 1.0.1 FASTX-toolkit by Assaf Gordon), removal of adapter sequences (3' adapter sequence (TGGAATTCTCGGGTGCCAAGG)). PCR duplicates were removed using the four random nucleotides on each side of the RNA fragment introduced with the adapters as molecular codes by running the tool “Collapse”. Then, the four random bases were removed from each side and “Collapse” was again used to obtain the unique sequences and the corresponding read counts corrected for PCR duplicates. Subsequently, a count table for all obtained unique sequences corresponding to a small ncRNA was generated using a series of further standard Galaxy tools as well as converting tools from the ToolShed (https://toolshed.g2.bx.psu.edu/). The resulting table contained the unique sequences and the number of reads per sample. CPM per sample filtering tool (Chen et al. 2014), removal of sequences with negligible read counts, comprising sequencing errors or sequences with very low evidence for potential expression, CPM cut-off: 8.41 cpm (corresponding to an average of 20 reads per library) for at least 3 out of 20 libraries. Small RNA sequences were annotated based on BLAST (Basic Local Alignment Search Tool) searches against a number of sequence databases. The collection of BLAST databases contained sequences from miRBase (bovine, human, and porcine precursor and canonical mature miRNAs, release 21), transcript sequences from NCBI and Ensembl, including non-coding RNAs, as well as tRNA and piRNA cluster sequences retrieved from the NCBI Bos taurus UMD 3.1.1 GFF3 file and http://www.smallrnagroup.uni-mainz.de/piRNAclusterDB.html , Rosenkranz D. piRNA cluster database: a web resource for piRNA producing loci. Nucleic Acids Research 2016 44(D1):D223-D230, respectively. Genome_build: bosTau8, UMD_3.1.1 Supplementary_files_format_and_content: *.txt: Tab-delimited text files: Supplementary_files_format_and_content: Filtered_sequence_counttable_smallRNA_exo.txt: Unique sequences and read counts per sample after filtering low abundance sequences. Supplementary_files_format_and_content: Filtered_sequence_counttable_smallRNA_exo_annotation_outliers_removed.txt: Unique sequences with annotation and read counts per sample, excluding some outlier samples. Supplementary_files_format_and_content: miRNA_isoforms_counts.txt: isomiR sequences and read counts per sample. Supplementary_files_format_and_content: miRNA_isoforms_summarized_counts.txt: isomiRs summarized read counts.
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Submission date |
Feb 09, 2018 |
Last update date |
Jul 31, 2018 |
Contact name |
Stefan Michael Bauersachs |
E-mail(s) |
stefan.bauersachs@uzh.ch
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Organization name |
University of Zurich
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Department |
Department for Farm Animals
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Lab |
Genetics and Functional Genomics
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Street address |
Eschikon 27 EHB 23.1
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City |
Lindau |
State/province |
Zurich |
ZIP/Postal code |
8315 |
Country |
Switzerland |
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Platform ID |
GPL19172 |
Series (2) |
GSE110443 |
Analysis of the small non-coding RNA content of bovine oviductal extracellular vesicles during the estrous cycle |
GSE110444 |
Analysis of the mRNA and small non-coding RNA content of bovine oviductal extracellular vesicles during the estrous cycle |
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Relations |
BioSample |
SAMN08514582 |
SRA |
SRX3679063 |
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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