|
| Status |
Public on Nov 27, 2019 |
| Title |
tfap2b_KO_RNA_R1 |
| Sample type |
SRA |
| |
|
| Source name |
tfap2b_KO_RNA_R1
|
| Organism |
Gallus gallus |
| Characteristics |
strain: Wild-type
development: Embryo
development: HH18
tissue: Neural crest (Cit+ cells)
isolation_method: Dissociation and FACS
treatment: pcU6:tfap2b_g1, pcU6:tfap2b_g2, pCAG Cas9-2A-Citrine
amplification: Illumina Nextera XT library preparation kit
assay: RNA-seq
|
| Treatment protocol |
Dissected vagal regions from electroporated embryos were dissociated with dispase (1.5mg/ml in DMEM/10mM Hepes pH 7.5) at 37°C for 15min with intermittent pipetting to achieve a single cell suspension and 0.05% Trypsin at 37°C for a final 3min. The reaction was stopped and cells were resuspended in an excess of Hanks buffer. Cells were centrifuged at 500g for 10min and resuspended in Hanks buffer, passed through a 0.22mm filter and centrifuged at 750g for 10min, pelleted cells were resuspended in 500µl Hanks buffer. Fluorescent positive cells were sorted and collected using BD FACS-Aria Fusion.
|
| Growth protocol |
Fertilised wild-type chicken eggs were obtained from Henry Stewart & Co (Norfolk), staged according to Hamburger and Hamilton (1951) (24). All experiments were performed on chicken embryos younger than 12 days of development, and as such were not regulated by the Animals (Scientific Procedures) Act 1986.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from dissected half dorsal neural tubes using Ambion RNAqueous Micro Total RNA isolation kit (Cat.#AM1931, ThermoFisher Scientific), the integrity was checked using Bioanalyser, and only samples with RIN>6 were used for analysis.
RNA-seq libraries were prepared using SMART-Seq™ v4 Ultra™ Low Input RNA Kit for Sequencing (Cat.#634889, Takara Bio Clontech) and sequenced using 40bp paired-end reads on Illumina NextSeq500.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Wild-type
RNA_featurecounts_KO.txt
|
| Data processing |
Reads were mapped using STAR (v.2.4.2a) for RNA-seq to galGal5. Read counts were obtained using subread (v.1.4.5) FeatureCounts
Differential Expression analysis was performed using DESeq2 R package.
Genome_build: galGal5
|
| |
|
| Submission date |
Jan 27, 2019 |
| Last update date |
Nov 27, 2019 |
| Contact name |
Tatjana Sauka-Spengler |
| E-mail(s) |
[email protected]
|
| Organization name |
MRC Weatherall Institute of Molecular Medicine
|
| Department |
University of Oxford
|
| Lab |
Sauka-Spengler lab
|
| Street address |
Radcliffe Department of Medicine
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19787 |
| Series (2) |
| GSE125710 |
Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages [CRISPR RNA-seq] |
| GSE125711 |
Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages |
|
| Relations |
| BioSample |
SAMN10826922 |
| SRA |
SRX5296694 |
