|
| Status |
Public on Sep 11, 2021 |
| Title |
B168: dsGFP_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Ixodes scapularis embryonic 6 (ISE6) cells
|
| Organism |
Ixodes scapularis |
| Characteristics |
cell line: Ixodes scapularis embryonic 6 (ISE6) cells
treatment: transfection with dsGFP
|
| Treatment protocol |
The dsRNA transfection was performed using Effectene (QIAGEN). 400ng dsRNA was diluted in in 100ul Buffer EC and 3.2ul Enhancer was added. The mixture was incubated for 2-5min at room temperature. Then, 10ul Effectene was added and incubated for 5-10min at room temperature. The mixture was added to the culture and the cells were incubated for 7-10 days. After the first incubation, the dsRNA transfection procedure was repeated again and incubated for 7-10 days to ensure the maximal efficacy of RNAi.
|
| Growth protocol |
Ixodes scapularis embryonic 6 (ISE6) cells were obtained from ATCC and cultured according to the published protocol at 34 degrees C (Munderloh and Kurtti, 1989). Cells were seeded at 1x10^6 /ml in 2ml fresh L-15B medium on a 6-well plate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from ISE6 cells using Trizol-LS (Invitrogen) according to the manufacturer’s instructions.For small RNA libraries of ISE6 cells depleted of small RNA factors and their control samples, 1ug total RNA was used for library construction. For 5’-tri-P libraries, the small RNA fraction (~15-35nt) was isolated by gel extraction and RNA species bearing 5’-OH were monophosphorylated by T4 polynucletide kinase (NEB). This was followed by treatment by a terminator exonuclease (Epicentre), dephosphorylation by Calf Intestine Phosphatase (NEB) and re-phosphorylation by T4 polynucletide kinase (NEB). For the oxidized small RNA library, small RNAs (~15-35nt) from 50ug total RNA was isolated by gel extraction and treated with 25mM NaIO4 dissolved in 60mM Borax buffer and incubated for 30 minutes at room temperature in the dark. For total RNAseq analysis, three sets of knockdown experiments were performed independently, and total RNA samples extracted by Trizol-LS (Invitrogen) were sent to BGI (Hongkong) for ribosomal RNA depletion using Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Epicentre).
For small RNA libraries of ISE6 cells depleted of small RNA factors and their control samples, 1ug total RNA was used for library construction using the TruSeq Small RNA Library Preparation kit (Illumina). Both 5’-tir-P enriched and oxidized samples were subjected to a small RNA library construction by the TruSeq Small RNA Library Preparation kit (Illumina). For total RNAseq analysis, library construction used non-stranded (Replicate 1) or stranded (Replicates 2 and 3) TruSeq mRNA Library Prep Kit (Illumina).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Total RNAseq data, library construction using non-stranded TruSeq mRNA Library Prep Kit (Illumina), sequencing on Hiseq4000.
|
| Data processing |
The adaptor sequences were trimmed using Cutadapt (version 1.18) with the default quality cutoff value (20).
Gene expression was quantified by salmon (v1.5.0) using the Vectorbase IscaI1.0 annotation
Differential gene expression analysis was done using DESeq2 package (version 1.26.0), with cut-off adjusted-p value set to 0.05.
genome build: Vectorbase IscaI1.0 annotation
processed data files format and content: The file "Processed_data_file_TotalRNASeq.xlsx" contains the results of DGE analysis using the total RNAseq libraries by the Salmon-DESeq2 pipeline.Sheet1: Ago-16 KD vs GFP control, Sheet2 RdRP1 KD vs GFP control, Sheet3: RdRP3 KD vs GFP control, Sheet4: TPM values for individual libraries.
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| |
|
| Submission date |
Sep 09, 2021 |
| Last update date |
Sep 11, 2021 |
| Contact name |
Katsutomo Okamura |
| Organization name |
Nara Institute of Science and Technology
|
| Street address |
8916-5 Takayama, Ikoma, NARA 630-0192 JAPAN
|
| City |
NARA |
| ZIP/Postal code |
630-0192 |
| Country |
Japan |
| |
|
| Platform ID |
GPL30611 |
| Series (1) |
| GSE183810 |
RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes |
|
| Relations |
| BioSample |
SAMN21369146 |
| SRA |
SRX12123217 |
