Format

Send to:

Choose Destination

SRX12123224: GSM5571684: B177: dsRdRP3_rep2; Ixodes scapularis; RNA-Seq
1 ILLUMINA (Illumina HiSeq 4000) run: 72.1M spots, 14.4G bases, 5.2Gb downloads

Submitted by: NCBI (GEO)
Study: RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes
show Abstracthide Abstract
Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) play essential roles in sRNA biogenesis in nematodes, plants and fungi, their involvement in other animals remains controversial. We identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs) expressed in the tick ISE6 cell line. The RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Knockdown of AGO/RdRP causes miregulation of protein-coding genes.These results demonstrate that arachnid RdRPs are important sRNA biogenesis factors, and the discovery of novel pathways underscores the importance of characterizing sRNA biogenesis in various organisms to understand virus-vector interactions and exploit RNAi for pest control. Overall design: For small RNA analysis, 9 small RNA libraries of ISE6 cells depleted of small RNA factors and 1 control sample were used. To study the chemical structures of 5' and 3' terminal nucleotides of tick sRNAs, oxidized sRNA library and 5'TriP enriched library were used. For total RNAseq analysis, three sets of knockdown experiments(knock down Ago-16, RdRP1 or RdRP3) were performed independently, with one set of control.
Sample: B177: dsRdRP3_rep2
SAMN21369159 • SRS10103480 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction 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).
Experiment attributes:
GEO Accession: GSM5571684
Links:
Runs: 1 run, 72.1M spots, 14.4G bases, 5.2Gb
Run# of Spots# of BasesSizePublished
SRR1583159172,063,95614.4G5.2Gb2021-09-12

ID:
16042599

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Support Center