|
| Status |
Public on Sep 11, 2021 |
| Title |
B057: dsRdRP1 |
| 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 dsRdRP1
|
| 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).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
small RNA library, sequencing on a Hiseq2000.
|
| Data processing |
Adaptor sequences were removed by using fastx_clipper(Part of FASTX Toolkit 0.0.14) and collapsed by fastx_collapser(Part of FASTX Toolkit 0.0.14) and converted by fasta_formatter(Part of FASTX Toolkit 0.0.14).
The fasta files were used for mapping using the ISE6 genome sequence (IscaI1) (Miller et al., 2018) using bowtie1.3.0 without allowing any mismatches.
For identification of sRNA sources, genome-mapping reads were mapped to the following reference sequences: 1. miRNAs (miRbase Release 22.1) (Kozomara et al., 2019), RNAP III transcripts (downloaded from RNAcentral) (Sweeney et al., 2021), rRNAs from NCBI and mRNAs (Vectorbase IscaI1.0) (Miller et al., 2018).
Python scripts were used to quantify reads that belong to different categories.
R script was used to normalize the data with RPM.
genome build: IscaI1
processed data files format and content: The file "Processed_data_file_sRNA_category.xlsx" contains summary of sRNA analysis:
processed data files format and content: Sheets1-13: sRNA read counts for each category. Each sheet report the numbers in each of the libraries.
processed data files format and content: Sheet14: Normalized counts of sRNAseq and total RNAseq reads mapping to the persistently present viruses.
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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 |
GPL18496 |
| Series (1) |
| GSE183810 |
RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes |
|
| Relations |
| BioSample |
SAMN21369137 |
| SRA |
SRX12123212 |
