|
| Status |
Public on Feb 04, 2022 |
| Title |
Krt V12_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
epithelial
|
| Organism |
Danio rerio |
| Characteristics |
age: 3dpf
batch: 2
genotype: mutant (HRas G12V)
cell type: keratinocyte
cell type id: CL:0000312
|
| Treatment protocol |
Zebrafish larvae were microinjected into one cell stage embryos and screened 3 days post fertilization for transgene expression. Microinjections for TRAP-RNA-sequencing included either control pTol2-krt4-RFP-HRas or oncogenic pTol2-krt4-RFP-HRasG12V constructs injected with pTol2-krt4-GFP-L10a in wildtype larvae, or into Tg(LyzC:EGFP-L10a) or Tg(mpeg1:EGFP-L10a) larvae for keratinocyte, neutrophil, or macrophage-specific expression profiling, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Larvae were stored at -80°C in minimal residual media. QIAshredder (Quiagen) was used to homogenize the larvae prior to immunoprecipitation. mRNA was isolated from immunoprecipitated polysomes from 50 pooled oncogene expressing fish or controls using TRIzol reagent (Invitrogen).
RNA quality and concentration was assessed on Agilent RNA PicoChip and samples with a concentration < 200 pg/µl were concentrated in a SpeedVac. As RNA concentrations were low, library prep was done with the NuGEN Ovation Single Cell RNA-Seq System with 20 cycles of amplification. Libraries were then checked by QuBit and an AATI Fragment Analyzer for concentration and fragment size. Adaptors with barcodes were used and samples were sequenced on an Illumina HiSeq with an average of 8 samples per lane, following manufacturer’s protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
polysomal RNA
|
| Data processing |
Reads were aligned to the transcriptome using Bowtie v1.1.1
Gene abundances were estimated using RSEM v1.2.20
Genome_build: GRCz10,Ensembl 83 annotation
Supplementary_files_format_and_content: The processed data file for each sample is the gene-level abundance output of RSEM and is a tab-delimited text file
|
| |
|
| Submission date |
Jan 13, 2022 |
| Last update date |
Feb 04, 2022 |
| Contact name |
Colin Dewey |
| Organization name |
University of Wisconsin-Madison
|
| Street address |
425 Henry Mall, 5420 Genetics/Biotechnology Center
|
| City |
Madison |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL14875 |
| Series (1) |
| GSE193591 |
Cell type specific transcriptome profiling reveals a role for thioredoxin in neutrophils and tumor initiation |
|
| Relations |
| BioSample |
SAMN24918785 |
| SRA |
SRX13773737 |
