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| Status |
Public on Dec 20, 2019 |
| Title |
1: EXP1-EBM2_smallRNA |
| Sample type |
SRA |
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| Source name |
BiologicalReplicate1.EBM_smallRNA
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Brain Microvascular Endothelial Cell
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| Treatment protocol |
EBM_only
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| Growth protocol |
HBMVECs (500,000/well) were cultured on Matrigel™-coated (BD Matrigel™ 10mg/mL, BD Biosciences, Franklin Lakes, NJ, USA) wells in a 6-well plate in endothelial basal medium (EBM-2)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for this study was isolated from samples using the Qiagen (Exiqon) miRCURY RNA isolation kit.
Small RNAseq libraries were generated using the NEB Next SmallRNA library preparation kit according to the manufacturer’s protocol, with some minor modifications. The reaction volumes were reduced to 1/5thof the recommended volume. Due to the low amounts of RNA input, the 3’ SR adaptor, SR RT primer and the 5’ SR adaptors were diluted 1:6 to avoid excessive amounts of free adaptors and the formation of adaptor dimers. The final libraries were purified using the Zymo DNA clean and concentrator-5 kit and subjected to size selection on the Pippin Prep with a cut off between 117 and 135 bp to deplete adaptor dimers and most of larger RNA species such as tRNA fragments.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
base-calling: The size selected libraries were sequenced on a HiSeq 4000 sequencing system.
read quality filtering: Condition2 Replicate1 was removed from analysis due to a markedly lower total miRNA count (273,468) compared to the other 8 samples (nearly 5x fewer counts than the sample with the next fewest counts). In the remaining samples, the samples with the fewest total miRNA counts (which was Condition3 Replicate2) had a total miRNA count of 1,338,649. Therefore, the scaled data for the remaining samples were filtered to remove miRNAs with fewer than 7.5 scaled counts (corresponding to a cutoff of 10 raw counts/1,338,649 total miRNA counts) in at least 2 samples; after this filtering, 430 miRNAs remained.
alignment: Paired-reads were mapped simultaneously to the GRCh37.83 cDNA and ncRNA transcriptomes (Kinsella et.al., 2011) using Kallisto[v0.44.0] (Bray et. al., 2016). Kallistos were imported to R with tximport. All reads mapping to ncRNA were excluded from the analysis, then transcript abundances were aggregated by Ensembl Gene ID. Genes with less than 10 reads across all samples were excluded from further analysis.
The data were further filtered using the Qlucore data analysis and visualization software package (www.qlucore.com) by selecting for miRNAs that displayed a variance across the dataset of 0.01, leaving 29 miRNAs. Differential expression analysis was then performed among the three conditions, and using a p-value <0.03/q-value <0.75, obtained 15 miRNAs.
Genome_build: GRCh37
Supplementary_files_format_and_content: tab delimited, transcript abundance fold change, p-value
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| Submission date |
Sep 27, 2019 |
| Last update date |
Dec 20, 2019 |
| Contact name |
Aleksandar Milosavljevic |
| E-mail(s) |
amilosav@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Department |
Genetics and Genomics
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| Lab |
Bioinformatics Research Laboratory
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE138114 |
Glioma-derived miRNA-containing extracellular vesicles induce angiogenesis by reprogramming brain endothelial cells (miRNA-seq) |
| GSE138115 |
Glioma-derived miRNA-containing extracellular vesicles induce angiogenesis by reprogramming brain endothelial cells |
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| Relations |
| BioSample |
SAMN12863623 |
| SRA |
SRX6917326 |
| 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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