|
| Status |
Public on Jan 15, 2023 |
| Title |
CF_2_3 |
| Sample type |
SRA |
| |
|
| Source name |
Immortalized Fibroblasts
|
| Organism |
Gallus gallus |
| Characteristics |
tissue: Fibroblasts
cell type: Immortalized Fibroblasts
breed: Broiler Ross 308
|
| Treatment protocol |
Immortalized chicken fibroblasts were seeded at a density of 4103 cells/cm2 or 0.3106 cells/mL for adherent and suspension cells, respectively. Transdifferentiation medium was composed of DMEM10 containing 200 µM oleic acid supplemented with 12 µg/mL L--Phosphatidylcholine (soy lecithin). Transdifferentiation medium was replaced every 2 d for 7 d. Cells were split on day 2 and reseeded back at starting cell density.
|
| Growth protocol |
Primary fibroblasts were passaged regularly, recording changes in doubling time and morphology as cells underwent senescence (crisis) and breakthrough (immortalization). Cells were considered to be immortalized once the culture showed stable morphology and doubling time following at least 120 population doublings from isolation. Immortalized lines were expanded and cryopreserved in four immortalized-cell banks labeled HUN-CF-1 to 4. Suspension culture of immortalized chicken fibroblasts Fibroblasts spheroids were created using Aggrewell 400 according to the manufacturer’s directions. Briefly, 1.2x106 spontaneously immortalized fibroblasts were seeded in DMEM with 10% FBS at a density of 3,867 cells per microwell for 48 hrs. Spheroids were then gently detached and transferred to a low-attachment 10 cm petri dish, in culture media supplemented with 0.1% Pluronic F-68. Spheroids were mechanically disturbed daily for 4 consecutive days by vigorous pipetting. Spheroids were transferred to baffled shaker flasks on day 7 and cultured in a humified shaker incubator at 39°C, 5% CO2 and 80 RPM in DMEM10 culture media supplemented with 0.1% Pluronic F-68. Large cellular aggregates were broken down by TrypLE™ digestion each week, and cells reseeded at a density of 200,000 cells/mL. Suspension cultures were counted and diluted back to initial density 2-3 times a week. Following 30 days of culture, the shaker speed was increased to 100 RPM leading to aggregate breakdown and single-cell suspensions developing for 60 days. Cells were considered anchorage-independent once culture showed stable doubling time and viability above 94%. Anchorage-independent cell lines were expanded and cryopreserved in two master-cell banks labeled FMT-SCF-2 and FMT-SCF-4 showing a doubling time of 192 and 202 hrs, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from primary and anchorage-independent cell lines using RNeasy Micro kit (Qiagen, USA) according to the manufacturer’s directions.
Library preparation and RNA sequencing were performed by Syntezza Bioscience (Israel). Library construction was conducted using NuGEN TrioRNA-Seq kit (Tecan, USA) and sequenced on Illumina HiSeqX with paired-end, 150 bp reads using the High Output V2 Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
expr.chicken.new.txt
|
| Data processing |
Sequencing reads were mapped to the UCSC chicken transcriptome (genome build galGal6) using Bowtie2 (22388286)
expression levels of all genes were quantified using RSEM (21816040).
Differential expression analysis was performed using R package DESeq2 (25516281) using default parameters. Reported are p-values from a negative binomial Wald-test. Overall, 3,641 and 3,739 genes showed significant differential mRNA expression between each primary and immortalized chicken cell line with BH-FDR<0.001, for Israeli Baladi and Broiler Ross breeds, respectively.
Genome_build: UCSC chicken transcriptome (genome build galGal6)
Supplementary_files_format_and_content: RSEM-expression matrix of inferred gene counts. (.txt); Differential expression analysis of primary and immortalized lines (.csv)
Assembly: galGal6
|
| |
|
| Submission date |
Dec 06, 2022 |
| Last update date |
Jan 15, 2023 |
| Contact name |
Avner Ehrlich |
| E-mail(s) |
avner.ehrlich@mail.huji.ac.il
|
| Phone |
972-54-3181422
|
| Organization name |
The Hebrew University of Jerusalem
|
| Department |
Bioengineering
|
| Lab |
MicroLiver Technologies Lab (Nahmias)
|
| Street address |
Silberman 3-512, Edmond J. Safra Campus (Givat Ram)
|
| City |
Jerusalem |
| ZIP/Postal code |
9190401 |
| Country |
Israel |
| |
|
| Platform ID |
GPL19005 |
| Series (1) |
| GSE169291 |
GM-free immortalization and high yield production of cultured chicken [RNA-seq] |
|
| Relations |
| BioSample |
SAMN32074360 |
| SRA |
SRX18511773 |
