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| Status |
Public on Nov 07, 2016 |
| Title |
HH14_RNAseq_rep1 |
| Sample type |
SRA |
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| Source name |
Spinal neural tube (brachial level)
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| Organism |
Gallus gallus |
| Characteristics |
strain: White Leghorn
tissue: Spinal neural tube (brachial level)
developmental stage: HH14
genotype: WT
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| Growth protocol |
Fertilized chicken eggs (White Leghorn, Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main, Dieburg). Incubation was done at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). After following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of Hamburger Hamilton (HH) (Hamburger and Hamilton, 1992).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Fertile chicken eggs were incubated at 37°C for 48 hrs until they reached stage HH14(Hamburger and Hamilton, 1992). Embryos were isolated from the eggs, transferred into 20 ml of 1xPBS and the extraembryonic membranes were removed. Under the stereomicroscope, the transverse SNT sections from chicken embryos were collected at the brachial level, extending caudally into the thoracic region (Figure 1C). Most of the tissues that are located laterally/ventrally to the neural tube (e.g. lateral plate mesoderm, endoderm and aorta) were removed manually. Each section was immersed in 90 mm petri dish, containing 3 ml of warm (37°C) trypsin (Trypsin.EDTA solution 1:250 (IX), GIBCO) (Figure 1C). Trypsinization time was tightly controlled to avoid over-digestion and dispersion of neural tissue and to retain the structural integrity of the SNT. Once connective tissues became loosened near the outline of the neural tube, which is easily detectable under a stereomicroscope, trypsin was inactivated by rinsing with cold (4°C) DMEM containing 10% FBS and samples were transferred into cold 1 x PBS. Following trypsin treatment, mesenchymal and ectodermal tissues (e.g. paraxial mesoderm, notochord, surface ectoderm) were removed manually using forceps. After isolation, SNT fragments were pooled and total RNA was isolated using Tripure reagent according to the manufacturer’s instructions (Roche). Libraries were prepared with the RNA sample preparation kit (TruSeq v2; Ilumina).
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the chicken reference assembly, version galGal10, using TopHat2
Gene expression for the individual samples was calculated as TPM using custom R scripts adapted from previous reports (Wagner et al., 2012).
For calculating the expression fold changes between HH19 and HH14 SNT, we performed a differential gene expression analysis of count data using custom R script and DESeq2 (Love et al., 2014).
Genome_build: galGal4
Supplementary_files_format_and_content: Txt file containing TPMs for each ENSEMBL chicken gene.
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| Submission date |
Nov 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Milos Nikolic |
| E-mail(s) |
milosn@gmail.com
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| Organization name |
Center for Molecular Medicine Cologne
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| Street address |
Robert Koch Str. 21
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| City |
Koeln |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50674 |
| Country |
Germany |
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| Platform ID |
GPL19005 |
| Series (2) |
| GSE89605 |
Epigenomic-based identification of major cell identity regulators within heterogeneous cell populations [RNA-seq] |
| GSE89606 |
Epigenomic-based identification of major cell identity regulators within heterogeneous cell populations |
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| Relations |
| BioSample |
SAMN05990958 |
| SRA |
SRX2333014 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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