|
| Status |
Public on Sep 29, 2020 |
| Title |
8dpi Swine Remote Zone 1 |
| Sample type |
SRA |
| |
|
| Source name |
Cardiac Tissue
|
| Organism |
Sus scrofa |
| Characteristics |
condition: 8dpi
cell type: Cardiac cells
other details: Remote Zone
|
| Growth protocol |
Primary cell isolates from reporter mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse cardiac interstitial cells (CIC) were obtained by enzymatic digestion. Erythrocytes were removed using RBC lysis buffer. A positive selection of CIC, enriched in Cardiac fibroblasts, was performed using LS columns and Feeder Removal MicroBeads (Miltenyi) according to the manufacturer’s instructions.
Bulk RNA-seq was performed following MARS-seq adapted for bulk RNA-seq with minor modifications. Briefly, poly-A RNA was extracted with Dynabeads Oligo dT (Ambion) and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) using poly-dT oligos (IDT) carrying a 7-bp index. Up to 8 samples with similar overall RNA content were pooled together and subjected to linear amplification via IVT using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs (NEB)). The resulting antisense RNA was fragmented into 250-350 bps fragments with RNA Fragmentation Reagents (Ambion) and dephosphorylated with 1U FastAP (Thermo Scientific) for 15 min at 37 °C. Partial Illumina adaptor sequences were ligated with T4 RNA Ligase 1 (NEB) followed by a second reverse transcription. Full Illumina adaptor sequences and a secondary barcode were added during library amplification with KAPA HiFi DNA Polymerase (Kapa Biosystems). Libraries were quantified using a Qubit 3.0 Fluorometer and their size profiles examined in Agilent’s 4200 TapeStation System. For Col1a1-GFP+ reporter mouse GFP+/CD31-/CD45- cardiac fibroblasts (CF), GFP-/CD31+/CD45- endothelial cells and GFP-/CD31-/CD45+ bone marrow-derived cells were FACS-sorted in Lysis/Binding Buffer (Ambion).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
8dpi_RemoteZone_SusScrofa_1
3' RNA-seq
Total RNA was isolated using TRIzol reagent after mechanical homogenization with an Ultra-turrax according to manufacturer’s instructions. RNA concentration was quantified using a Qubit 3.0 Fluorometer and its quality was examined in Agilent’s 4200 TapeStation System.
processed data file: SusScrofa_Counts.txt
8dpi_RemoteZone_SusScrofa_1.fastq.gz
|
| Data processing |
bcl2fastq was used to demultiplex .bcl files and retrieve fastq files according to the sample barcodes.
Fastq files were aligned to the corresponding genome (mm10, GRCh38, Sscrofa 11.1) using STAR (v 2.6.1).
Gene level quantification was performed using Rsubread and ensembl transcriptomes (GRCm38.91, Sscrofa11.1.93, and GRCh38.92).
Normalization, processing and differential expression analyses were performed using DEseq2.
Genome_build: Mouse mm10 (GRCm38), Human GRCh38, Pig Sscrofa 11.1
Supplementary_files_format_and_content: *_Counts.txt: Files with raw counts after gene level quantification.
|
| |
|
| Submission date |
Jun 03, 2019 |
| Last update date |
May 03, 2024 |
| Contact name |
Silvia C Hernandez |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
9295049664
|
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Medicine- Cardiology
|
| Lab |
Frangogiannis
|
| Street address |
1300 Morris park avenue
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL20983 |
| Series (2) |
| GSE132143 |
Single cell RNA-seq analysis reveals the crucial role of Collagen triple helix repeat containing 1 (Cthrc1) cardiac fibroblasts for ventricular remodeling after myocardial infarction [RNA-seq] |
| GSE132146 |
Single cell RNA-seq analysis reveals the crucial role of Collagen triple helix repeat containing 1 (Cthrc1) cardiac fibroblasts for ventricular remodeling after myocardial infarction |
|
| Relations |
| BioSample |
SAMN11949966 |
| SRA |
SRX5962149 |
