|
| Status |
Public on Sep 28, 2023 |
| Title |
RNA_amPA-M_rep4 |
| Sample type |
SRA |
| |
|
| Source name |
polyA RNA-seq in Pcca-/- male 8-week-old mouse heart
|
| Organism |
Mus musculus |
| Characteristics |
tissue: heart
genotype: FVB Pcca-/- A138T mutation
age: 8-week-old
|
| Treatment protocol |
To harvest tissues, 8-week-old mice were killed humanely by i.p. injection of an appropriate volume adjusted for body weight of sodium pentobarbitone (stock 200 mg/ml) pre-mixed at a 2:1 ratio with sodium heparin (stock 5,000 I.U./mL).
|
| Growth protocol |
Mice were bred and housed in individually ventilated cages and all animals had ad libitum access to water and food (Teklad #2918 global 18% protein rodent diet). Mice used were either wild-types (Pcca+/+ A138T; amWT) for control experiments, or homozygous (Pcca-/- A138T; amPA). Only heterozygous or wild-type dams were used for breeding and genotyping by PCR was performed.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Ventricular tissue from 8-week mice was snap-frozen and cryoground. Bulk RNA was extracted using the Zymo Quick-RNA Miniprep Plus Kit (R1057), following the manufacturer’s protocol with some modifications in the tissue digestion stage. All centrifugation steps were performed at 16,000 RCF at 4°C. 20 mg of cryoground tissue was mechanically homogenised on ice with disposable probes (Qiagen TissueRuptor II) in 350 µL of DNA/RNA Shield. 45 µL of Proteinase K mix was added to 300 µL of sample, incubated for 30 mins at 55°C, and centrifuged for 2 mins after incubation. The supernatant was transferred to new microcentrifuge tubes and 300 µL RNA Lysis Buffer was added at 1:1 to the supernatant. Thereafter, the RNA purification procedure followed the manufacturer’s protocol, with some differences: half volume ethanol (300 µL) was added, in order to isolate RNAs ≥ 200 nt for poly(A) enrichment; DNase I treatment was performed in-column; RNA was eluted in non-DEPC-treated nuclease-free water.
PolyA RNA was purified using the NEBNext Poly(A) mRNA magnetic isolation module (E7490) and DNA libraries were prepared using the NEBNext Ultra II Directional RNA library preparation kit for Illumina (E7765).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
RNA-seq reads were aligned to the mm10 genome using STAR
Duplicate reads were removed using samtools rmdup
Read counts were determined using featureCounts (Subread package)
Differential gene expression analysis was conducted using DESeq2
Assembly: mm10
|
| |
|
| Submission date |
Jun 10, 2022 |
| Last update date |
Sep 28, 2023 |
| Contact name |
Pawel Swietach |
| Organization name |
University of Oxford
|
| Department |
Department of Physiology, Anatomy & Genetics
|
| Street address |
Sherrington Building, Parks Road
|
| City |
Oxford |
| ZIP/Postal code |
OX1 3PT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE205834 |
Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation [RNA-Seq 1] |
| GSE205838 |
Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation |
|
| Relations |
| BioSample |
SAMN28963068 |
| SRA |
SRX15672417 |
