|
| Status |
Public on Mar 20, 2024 |
| Title |
MCC progenitors, Wildtype, Stage 26 Exp2 |
| Sample type |
SRA |
| |
|
| Source name |
MCC progenitors
|
| Organism |
Xenopus laevis |
| Characteristics |
genotype: Wildtype
Stage: Stage 26
cell type: MCC progenitors
replicate: 2
|
| Treatment protocol |
Both wildtype and emi2 mutant embryos were injected at the 2-4 cell stage with an inducible multicilin (multicilin fused to the human glucocorticord receptor ligand binding domain). At stage 10, skin progenitors were isolated (40 animal caps/sample) and treated with dexmethasone at stage 11 to induce multicilin activity, which converts most if not all of the cells into MCC progenitors.
|
| Growth protocol |
Fertilized Xenopus eggs were injected with Cas9 protein along with a gRNA directed agains Emi2 to generate FO mutants.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the equivalent of stage 17 and stage 26, total RNA was extracted using the proteinaseK method, followed by phenol:chloroform extraction, precipitation in 4M LiCl, resuspension in buffer, phenol-chloroform extraction, and ethanol precipitation.
RNAseq libraries were constructed with Illumina Truseq RNA Sample Preparation kit v2 according to the manufacturer’s instructions and sequenced on a HiSeq 2500 at 1x50 base pairs to a depth of 20-40 million reads.
Each RNAseq condition was performed in triplicate, using animal caps isolated from different females on different days.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were quality-tested using FASTQC.
Reads were mapped to v2 of the Xenopus laevis 9.2 genome using RNA-STAR. Mapping was carried out using default parameters (up to 10 mismatches per read, and up to 9 multi-mapping locations per read).
The aligned reads were then counted, normalized, and tested for differential expression using the HOMER software package from UCSD and edgeR. Differentially expressed genes were defined as having a false discovery rate (FDR) <0.05 and a log2 fold change >1 when comparing two experimental conditions.
For visualization purposes, RNA-seq reads were also mapped to the Xenopus laevis genome with bowtie2 and loaded as tracks into the IGV browser.
Genome_build: Xenopus laevis 9.2
Supplementary_files_format_and_content: Excel table containing normalized counts and differential expression statistics.
|
| |
|
| Submission date |
Jan 28, 2022 |
| Last update date |
Mar 20, 2024 |
| Contact name |
April Elizabeth Williams |
| E-mail(s) |
apriljack06@gmail.com, awilliams@salk.edu
|
| Phone |
7345461645
|
| Organization name |
Salk Institute for Biological Studies
|
| Department |
IGC
|
| Street address |
10010 N Torrey Pines Rd
|
| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL18936 |
| Series (1) |
| GSE195635 |
RNAseq analysis of Xenopus laevis wildtype and emi2 mutant multiciliated cells during and post differentiation |
|
| Relations |
| BioSample |
SAMN25353220 |
| SRA |
SRX13966098 |
