|
| Status |
Public on Feb 06, 2015 |
| Title |
CreNeg (Isl1 fl/fl)-2 |
| Sample type |
SRA |
| |
|
| Source name |
heart - sinoatrial node
|
| Organism |
Mus musculus |
| Characteristics |
strain background: mixed
developmental stage: Embryonic Day 12.5
genotype/variation: Hcn4-GFP;Isl1 fl/fl
injected with: Tamoxifen at embryonic day 10.5
tissue: heart; sinoatrial node
|
| Treatment protocol |
Embryos or hearts were removed intact, washed with cold PBS, and immediately embedded in OCT and stored at -20 °C until sectioning. Tissue was sectioned at a thickness of 8 microns onto membrane-coated slides (MembraneSlide NF 1.0 PEN, Zeiss Microscopy, Gottingen, Germany). For laser capture, slides were thawed to room temperature until evaporation of moisture (approximately 1 minute), placed on the microscope stage of a PALM Micro-Beam inverted microscope with LCM capability (Zeiss). The sinus node tissue and right atrial tissue were identified visually and outlined manually with the microscope user interface (Online Video 2). Laser power and catapult energy were optimized prior to the experiment and varied from experiment-to-experiment. After each experiment, sections were stained with DAPI and anti-Hcn4, mounted, and visualized to confirm accurate dissection of the region of interest.
|
| Growth protocol |
Genetically modified mice were maintained on a mixed background.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from regions of interest isolated with laser capture using the PicoPure Kit (Arcturus) according to manufacturers instructions.
RNA-seq libraries were prepared with the Ovation RNA-Seq System V2 kit (NuGEN) according to manufacturers instructions. Briefly, total RNA is reverse-transcribed using a combination of random hexamers and a poly-T chimeric primer, followed by second strand synthesis using DNA polymerase. The cDNA was then amplified using single primer isothermal amplification (SPIA). Libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit (NuGEN) according to manufacturers instructions. The RNA-seq libraries were analyzed by Bioanalyzer (Agilent) and quantified by QPCR (KAPA).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
WT.2
|
| Data processing |
Short read assembly was performed using TopHat v2.0.6 against mm8 build of the genome.
Gene counts were determined using Rsamtools and sample normalization was performed using edgeR.
Differential expression was performed using edgeR.
Genome_build: mm8
Supplementary_files_format_and_content: text-tab delimited with rows as genes and columns as samples; contains normalized counts as output by edgeR
|
| |
|
| Submission date |
Feb 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Vasanth Vedantham |
| E-mail(s) |
vedanthamv@medicine.ucsf.edu
|
| Organization name |
University of California, San Francisco
|
| Department |
Medicine-Cardiology
|
| Street address |
505 Parnassus Avenue, M1176D
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE65658 |
Mouse sinoatrial node transcriptome |
|
| Relations |
| SRA |
SRX864595 |
| BioSample |
SAMN03328602 |
