|
| Status |
Public on Nov 30, 2020 |
| Title |
CpG sample 3 miRNA |
| Sample type |
SRA |
| |
|
| Source name |
bovine macrophage cell line
|
| Organism |
Bos taurus |
| Characteristics |
cell line: BoMac
cell type: peripheral blood monocyte
treatment: CpG
replicate: 3
|
| Treatment protocol |
BoMac cells were plated into 6-well plates at least 24 h before treatment. CpG or pI:C was later added at 50 ug/ml and 10 ug/ml, respectively. Cells were then incubated for 6 h and thereafter harvested for RNA isolation.
|
| Growth protocol |
BoMac cells were routinely cultured in IMDM medium supplemented with fetal bovine serum, antibiotic-antimycotic, non-essential amino acids, vitamins and beta 2 mercaptoethanol. Cells were subcultured every 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted with the Qiagen miRNeasy kit according to the manufacturers' instructions.
small RNA libraries were prepared using the TruSeq Small RNA Sample Prep kit (Illumina).
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
CpG sample 3
CpG_Expt33_GATCAG
|
| Data processing |
The 9 libraries were quantitated by qPCR, pooled in equimolar amounts and sequenced on one lane for 51 cycles from one end of the fragments on a HiSeq 4000 using a HiSeq 4000 sequencing kit version 1.
Fastq files were generated and demultiplexed with with the bcl2fastq v2.17.1.14 Conversion Software (Illumina) with -ml 10 option , which trims off adaptors and removes sequences less than 10 nt
Reads 17nt or longer were used to find known and novel miRNAs using miRDeep2 (Friedlander et al. 2012; Nucleic Acids Res, 40(1), 37-52. doi:10.1093/nar/gkr688) using mature and precursor sequences from miRBase release 21 for Bos taurus, Capra hircus and Ovis aries and also and the B. taurus reference genome (release UMD_3.1.1, NCBI Assembly ID GCF_000003055.6)
The resulting novel and known miRNA predictions from miRDeep2 were then checked for overlapping predicted regions. Any scores from predicted regions with an overlap were compared, and the region with the highest overall score in the comparison was retained to break the tie. These results were combined with the miRBase bovine-specific miRNAs that miRDeep2 reported as not being found in the reference genome.
Adapter-trimmed reads were initially de-replicated per sample, retaining counts and sample ID information in the sequence header. Per-sample de-replicated reads were then concatenated and aligned using Novoalign 3.06.03 (using the miRNA mode) to the miRDeep2 predicted mature and hairpin sequences for B. taurus. The final sorted alignments (now containing read IDs that have the sample and count) were then parsed by a simple script to generate accurate miRNA counts.
Data from counts mapping uniquely to mature miRNAs had the lowest level of ambiguity; these were used in downstream gene expression analyses.
Genome_build: NCBI UMD_3.1.1
Supplementary_files_format_and_content: counts
|
| |
|
| Submission date |
Feb 26, 2018 |
| Last update date |
Nov 30, 2020 |
| Contact name |
Felix Toka |
| E-mail(s) |
ftoka@rossvet.edu.kn
|
| Phone |
+7328980078
|
| Organization name |
Ross University School of Veterinary Medicine
|
| Department |
Biomedical Sciences
|
| Lab |
Centre for Integrative Mammalian Research
|
| Street address |
West Farm
|
| City |
Basseterre |
| ZIP/Postal code |
00334 |
| Country |
Dominica |
| |
|
| Platform ID |
GPL23295 |
| Series (1) |
| GSE111162 |
miRNA-Seq analysis of the early phase of BoMac cell stimulation with a viral and a bacterial pathogen associated molecular patterns |
|
| Relations |
| BioSample |
SAMN08611264 |
| SRA |
SRX3744330 |
