|
| Status |
Public on Sep 03, 2023 |
| Title |
PumKO2 biological rep1 |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: cell line
cell line: DL1
genotype: Pumilio knockout
|
| Treatment protocol |
For RNAi experiments, cells (3 million cells in 1 mL of serum free SDM per well of a 6-well dish) were bathed with 20 µg of dsRNA corresponding to either Pum, Not1, Pop2, or LacZ non-targeting control for 60 minutes. Then 2 mL of SDM containing 5% heat-inactivated FBS was added to each well. After 48 hours, the cells were harvested by centrifugation and suspended in 2 mL Phosphate Buffered Saline (PBS). RNA was purified from 1.5 mL of the cells, and the remaining 0.5 mL was reserved for western blot analysis.
|
| Growth protocol |
DL1 cells were grown in SDM media
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was purified using the Maxwell RSC simply RNA tissue extraction kit (Promega) with on-bead DNase I digestion. The RNA concentration was determined using a NanoDrop spectrophotometer. RNA samples were submitted for Agilent Tapestation analysis and RNA integrity number (RIN) values were 8.7-10. Total RNA was poly(A) selected using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolab).
We reverse transcribed 1 ug of total RNA with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. We click-ligated the p5 adapter (IDT) to the 5′ end of the cDNA with CuAAC. We then amplified the cDNA for 21 cycles with Universal primer and 3′ indexing primer and purified it on a 2% agarose gel by extracting amplicon from 200-300 base pairs. We pooled the libraries and sequenced single-end, 75 base-pair reads on a Nextseq 550 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
sequencing adapters were trimmed and unique molecular identifiers (UMIs) annotated using fastp (version 0.14.1)
reads were aligned to the UCSC dm6 genome using hisat2 (version 2.1.0).
The alignments were then deduplicated using UMI-tools (version 1.0.1) and differential expression was analyzed using DEseq2 (1.23.110) and featureCounts (Rsubreads 1.30.9).
Assembly: dm6
|
| |
|
| Submission date |
Aug 30, 2023 |
| Last update date |
Sep 03, 2023 |
| Contact name |
Aaron C Goldstrohm |
| E-mail(s) |
[email protected]
|
| Phone |
7349450789
|
| Organization name |
University of Minnesota
|
| Department |
Biochemistry, Molecular Biology, and Biophysics
|
| Lab |
6-155 Jackson Hall, 1214A
|
| Street address |
321 Church Street S.E.
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE241940 |
Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase |
|
| Relations |
| BioSample |
SAMN37199552 |
| SRA |
SRX21525937 |
