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Sample GSM4861803

Query DataSets for GSM4861803

Status

Public on Sep 22, 2021

Title

Uninfected replicate 3

Sample type

SRA

Source name

Chicken caecum

Organism

Gallus gallus

Characteristics

infection status: Uninfected
sample time post-infection [days]: 0
breed: Dekalb White Leghorn-type laying chicken/Bovans Robust

Treatment protocol

At 12 days of age blood samples were collected and analysed for maternal antibodies to E. tenella using an in house ELISA as previously described (Wattrang et al., 2016). Chickens were then allocated to six groups, n=6, with respect to an even distribution of maternal antibodies. One group was kept as uninfected controls and at 17 days of age chickens in the remaining five groups were inoculated orally with 1000 live E. tenella oocysts per bird. Viability of oocysts was estimated based on our experience that approx. 12 % of oocysts loose infectivity per month of storage in 2 % dichromate at 4 °C and oocysts used in this experiment had been stored for 3 months. From day 5 to day 9 (120 h to 218 h) after infection all faeces produced by chickens were collected and numbers of oocysts per gram faeces (OPG) were determined as described earlier (Wattrang et al., 2016; Wattrang et al., 2019).

Growth protocol

A pure E. tenella Houghton strain (Chapman & Shirley, 2003) isolate was maintained by twice yearly passage in chickens and sporulated oocysts were prepared as according to earlier described protocols (Wattrang et al., 2016). For this study, 36 female Dekalb White Leghorn-type laying chickens (in Sweden marketed under the name Bovans Robust) purchased from a commercial hatchery were used. An additional group of 6 chickens from the same batch not included in the present study were infected in parallel and data for oocyst excretion and caecal lesion scoring of these chickens are presented herein. All chickens were reared from day-old under SPF-conditions at the National Veterinary Institute animal facilities and were group housed in cages in rooms under negative pressure ventilation.

Extracted molecule

total RNA

Extraction protocol

At 1, 2, 3, 4 and 10 days after infection all chickens in one of the infected groups and one or two of the uninfected control chickens were killed by cervical dislocation and the two caeca were collected. Approximately 1 cm of the proximal part of the caeca containing the caecal tonsils was cut off. The remaining caeca were cut open longitudinally and caecal contents were thoroughly rinsed off with ice cold PBS. Ceacal tissues were cut into 0.5 cm wide strips and the tissue from the two caeca from each bird where pooled in 5 ml RNAlater (RNAlater® stabilization solution, Invitrogen, ThermoFisher Scientific). Samples in RNAlater were subsequently stored at 4°C for 24 h and thereafter stored at -20°C until RNA isolation. Caecal tissues stored in RNAlater were thawed, removed from the solution and briefly air-dried. Tissues were then added to test tubes containing 10 ml of TRIzol and a 1:1 mixture of 2 mm ø and 0.5 mm ø XX beads (XX). Tubes were placed in a XX and shaken three times for 60 s at 6.2 m/s. After this treatment all mucosal tissue but not all connective tissue was dissolved. Samples were then stored at – 70°C until RNA isolation. For RNA isolation 1 ml from each of the caecal samples homogenised in TRIzol was used and RNA was extracted using chloroform according to the TRIzol manufacturer’s RNA isolation protocol. The Isolated RNA was subsequently treated with DNase (TURBO™ DNase, 2U/ul, ThermoFisher Scientific) according to the manufacturers protocol (Pub. No. MAN0001271) and further purified using reagents and the “RNA cleanup” protocol of the RNeasy Mini kit (Qiagen) protocol. RNA concentration and quality was then assessed using the Agilent RNA 6000 Nano kit on a 2100 Bioanalyzer Instrument (Agilent) and the RNA stored at -70°C until further analysis.
Sequencing libraries were prepared from 500ng total RNA using the TruSeq stranded mRNA library preparation kit (Cat# 20020594/5, Illumina Inc.) including polyA selection.The library preparation was performed according to the manufacturers’ protocol (#1000000040498). NovaSeq 6000 S4 flowcell, paired-end 150bp read length, v1 sequencing chemistry.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

TC-2482-19
chicken_counts.csv
eimeria_counts.csv
chicken_counts_norm.csv

Data processing

Base calling with Illumina basecaller
Reads were trimmed to get rid of adapters and low-quality parts with Trimmomatic v. 0.36, using the standard Illumina adapter library, SLIDINGWINDOW:4:20 and MINLEN:50
Reads were mapped to a merged version of the reference genomes (effectively mapping to both at the same time as if they were a single genome) with STAR v. 2.7.2b, using default settings
Reads mapping to features were counted using HTSeq v. 0.9.1, with strandedness set on reverse, i.e. -s reverse, and otherwise default settings
Read counts loaded into R and, using edgeR, each sample was CPM normalized with regards to library size and genes filtered out that had few/no reads mapping to them across samples
Genome_build: GRCg6a/Eth001
Supplementary_files_format_and_content: comma separated text files containing raw gene counts for each gene and sample, one for the chicken data and one for E. tenella
Supplementary_files_format_and_content: comma separated text files containing CPM normalized read counts for each organism, with low expressed genes across time points filtered out along with samples without chicken data, for the chicken dataset, and samples without E. tenella data, for the E. tenella dataset

Submission date

Oct 26, 2020

Last update date

Sep 22, 2021

Contact name

Arnar Kari Sigurdarson Sandholt

E-mail(s)

a.k.sigurdarsonsandholt@uu.nl

Organization name

Utrecht University

Department

IRAS