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Sample GSM4618501

Query DataSets for GSM4618501

Status

Public on Aug 01, 2020

Title

R07

Sample type

SRA

Source name

Secretion product

Organism

Heligmosomoides bakeri

Characteristics

product type: Extracellular vesicle
developmental stage: Adult parasites

Treatment protocol

N/A

Growth protocol

T. muris were cultured for 22 hr in RPMI supplemented with 500 U/ml penicillin and 500 μg/ml, conditioned media was collected at 4 hr, discarded and replaced. Conditioned media from 4-22hr was collected, spun at 720 x g for 15 minutes and filtered through a 0.2μm filter. EVs were isolated using ultracentrifugation at 100,000 x g for 2 hr, and washed twice using PBS. EV pellets were re-suspendedin 6 ml of PBS and stored frozen until required. EVs concentrated to 185 μl using a 5kDa MWCO vivapspin and RNA extracted using Qiagen miRNeasy mini kit. Some samples were treated with either polyphosphatase, DNase or both. Small RNA libraries were prepared using CleanTag small RNA library preparation kit from Trilink using 18 PCR cycles. For H.bakeri EVs, the filtered HES were concentrated in a Vivaspin 20 MWCO 3kDa concentrator (Sartorius, Göttingen, Germany). 400 μL of HES (concentration 5mg/mL) was separated into 1 ml fractions by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) in PBS with an AKTA basic FPLC system (GE Healthcare). Libraries were prepared using the CleanTag small RNA Library prep kit (Trilink) following manufacturer’s instructions. Adapters were diluted 1:12 and 18 PCR cycles used. Quantification and size analysis of H. bakeri EVs was performed using the qNano Gold platform (Izon Science).

Extracted molecule

total RNA

Extraction protocol

Qiagen miRNeasy mini kit
CleanTag small RNA library prep kit, 18 PCR cycles

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

small RNAs
Hb_EvU_Un_1

Data processing

Sequence quality using FastQC
Adapters were removed using cutadapt
Genome alignments of sequences using bowtie to the host genome mus musculus (version GRCm38, with additional RefSeq rRNA, as all mouse rRNA genes are not present in the genome) and T. muris (GCA_000612645.2) requiring perfect matches.
miRNAs were predicted using miDeep2 v2.0.0.8 utilizing sequences from miRBase v22 and additional nematode miRNA sequences from Brugia pahangi.
RNA biotypes were identified using the genome version PRJEB126 TMUE3.0 downloaded from WormBase ParaSite, miRNA identified as above, rRNA using cmsearch from the infernal [v1.1.2], (Friedländer et al., 2012); rRNAs using cmsearch from the infernal [v1.1.2] (Nawrocki et al., 2013) suite to search for eukaryotic rRNAs using covariance models from Rfam [version 14.1], tRNAs using tRNA-scan-SE [v1.1.1], snRNA, protein-coding exons, and protein-coding introns were extracted from publicly available GFF3 annotation on WormBase ParaSite genome PREJEB126.

Submission date

Jun 16, 2020

Last update date

Aug 01, 2020

Contact name

Amy Buck

E-mail(s)

amybucklab@gmail.com, a.buck@ed.ac.uk, sujai.kumar@ed.ac.uk

Organization name

The University of Edinburgh

Department

IIIR