|
| Status |
Public on Mar 22, 2023 |
| Title |
SCF5 |
| Sample type |
SRA |
| |
|
| Source name |
Spontaneously Immortalized, anchorage-independent , chicken embryonic fibroblast
|
| Organism |
Gallus gallus |
| Characteristics |
breed: Israeli Baladi
cell type: Spontaneously Immortalized chicken embryonic fibroblast
cell type: anchorage-independent
|
| Treatment 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. 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 master-cell banks labeled FMT-SCF.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from FMT-SCF-1 to 5 using Quick-DNA™ MicroPrep Zymo-spin IC columns (ZR-D3021, Zymo research, Irvine CA) according to the manufacturer’s instructions. DNA was dehydrated on GenTegra DNA tubes and shipped to Syntezza (Jerusalem, Israel) for library preparation.
Library was construsted using Celero DNA library prep kit (Tecan Genomics), according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Library strategy: WGS
The DNA libraries were sequenced using Illumina’s NovaSeq 6000 using Novaseq S4 cartridge (Novogene, Singapore).
The average coverage was 25X and was uniformly distributed. Whole genome sequence reads were aligned to the chicken reference genome draft (version GRCg6a) using Bowtie2 (22388286) 40.
Segmental copy number variation and aneuploidy were predicted by AneuFinder with default parameters using two size bin length of 15Kbp and 1Mbp, respectively.
Heatmap and dendrograms were plotted using R.
Genome_build: chicken reference genome draft (version GRCg6a)
|
| |
|
| Submission date |
Jul 20, 2021 |
| Last update date |
Mar 22, 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 |
GPL26853 |
| Series (2) |
| GSE180441 |
GM-free immortalization and high yield production of cultured chicken [WGS] |
| GSE180442 |
GM-free immortalization and high yield production of cultured chicken |
|
| Relations |
| BioSample |
SAMN20325538 |
| SRA |
SRX11504063 |
