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Sample GSM5984213

Query DataSets for GSM5984213

Status

Public on Jan 20, 2023

Title

CAM11M_24

Sample type

SRA

Source name

Chorioallantoic membrane

Organism

Gallus gallus

Characteristics

breed: Ross 308 broiler
tissue: Chorioallantoic membrane
age: Prehatch : 11 days of incubation
gender: Male

Growth protocol

Fertilized eggs (ROSS 308) were stored 3 days (16°C, 80% relative humidity) prior to incubation during 11 or 15 days (55% Relative humidity, 37,8°C). After 11 or 15 days of incubation, eggs were opened and embryos were decapited following IACUC guidelines. A piece of embryo neck was removed to perform molecular sexing. Chorioallantoic membranes were removed from eggs, quickly rinsed in sterile physiologic solution and frozen in liquid nitrogen. Samples were all stored at -80°C prior to RNA extraction.

Extracted molecule

total RNA

Extraction protocol

RNA extraction was achieved using Macherey Nagel Nucleospin RNA kit. RNA concentration was determined using Nanodrop and RNA quality control was checked using Agilent. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

CAM11M_24

Data processing

Platform HiSeqPE-150
Clustering and sequencing The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.
Quality control Raw data (raw reads) of FASTQ format were firstly processed through in-house scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Data filtering Raw reads are filtered to remove reads with adapter contamination or reads with low quality (Discard reads with adaptor contamination;Discard reads when uncertain nucleotides constitute more than 10% of either read (N > 10%); Discard reads when low quality nucleotides (base quality less than 20) constitute more than 50% of the read)
Mapping to reference genome Reference genome (GRCg6a (GCA_000002315.5)) and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Paired-end clean reads were mapped to the reference genome using HISAT2 software. HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome. These small indexes (called local indexes), combined with several alignment strategies, enable rapid and accurate alignment of sequencing reads.
Quantification HTSeq was used to count the read numbers mapped of each gene, including known and novel genes. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. RPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Assembly: (GRCg6a (GCA_000002315.5))
Supplementary files format and content: tab-delimited text files include FPKM values for each sample

Submission date

Mar 30, 2022

Last update date

Jan 20, 2023

Contact name

Sophie REHAULT-GODBERT

E-mail(s)

sophie.rehault-godbert@inrae.fr

Organization name

INRAE

Street address

INRAE Centre Val de Loire, UMR BOA