|
| Status |
Public on Nov 21, 2022 |
| Title |
LHX2_KD_2 |
| Sample type |
SRA |
| |
|
| Source name |
hES-RPE
|
| Organism |
Homo sapiens |
| Characteristics |
strain: ES-derived RPE cells
genotype: LHX2_KD
age: 14d in differentiation conditions
|
| Treatment protocol |
The downregulation of Lhx2 was constructed using pGIPZ vectors (Thermo Fisher Scientific). Four different pGIPZ vectors containing four different shRNA sequences targeting Lhx2 mRNA (NM_004789) were examined for their downregulation efficiency using qRT-PCR. Two of the four vectors which showed the strongest effect were then used. A sh-non-silencing pGIPZ vector (RHS4531) was used as a negative control. To produce the infectious viruses, the 293T packaging cell line was co-transfected using jetPEI (Polyplus) with the lentiviral backbone plasmids shLhx2-pGIPZ or the sh-non-silencing-pGIPZ, packaging plasmid pCMVΔR8.2 and envelope plasmid pVSV-G. After 72hr, the virus particles in the medium were collected and filtrated through a 0.45 μm pore size filter. 2.5x10^5 hRPE-ES cells were seeded in 24 well plate and were grown for 14d to achieve full differentiation. For the infection, cells were incubated with virus-containing medium in the presence of 100 μg/ml polybrene (hexadimethrine bromide; Sigma) for 30min in 37°C followed by centrifugation (30min, 37°C, 1100xg). Medium was afterwards replaced with fresh growth medium. After 24hr 10 μg/ml puromycin (Sigma) was added for additional 5 days followed by harvesting for RNA isolation.
|
| Growth protocol |
For RPE differentiation, hESCs colonies (Hes1 hES) were picked up using collagenase IV (1 mg/ml; 200 U/mg, Gibco-BRL, Gaithersburg, MD), and cultured using differentiation protocol including treatment with nicotinamide and activinA. After 6-10 weeks in suspension, pigmented clusters were triturated, plated on gelatin (Sigma) coated plates (Corning Incorporated, Corning, NY), and cultured in Knock out medium with 10mM nicotinamide for 3-5 weeks. The RPE cell lines were further propagated using TrypLE Select (Gibco-BRL). The cells were passaged every 2 weeks.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using the QIAshredder and the RNeasy kits (QIAGEN)
Sequencing libraries were prepared using the INCPM mRNA protocol
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
LHX2_KD_RNAseq_Counts.xlsx
|
| Data processing |
Sequenced reads were mapped to the homo sepians genome (hg19), using TopHat v2.0.10
Genes were identified using a .gtf obtained from Ensembl release 82. Per gene reads were counted using HTSeq.
Normalization and detection of differentially expressed genes were done using DEseq2
Genome_build: hg19
Supplementary_files_format_and_content: Excel table with gene count
|
| |
|
| Submission date |
Jun 14, 2021 |
| Last update date |
Nov 21, 2022 |
| Contact name |
Mai Eshel |
| E-mail(s) |
maieshel@mail.tau.ac.il
|
| Organization name |
Tel Aviv University
|
| Department |
Human Molecular Genetics and Biochemistry
|
| Lab |
Elkon
|
| Street address |
Klatzkin 35
|
| City |
Tel Aviv |
| ZIP/Postal code |
6997801 |
| Country |
Israel |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE178163 |
RNA-seq analysis of hES-RPE cells in 14d from LHX2 KD and control - non-specific shRNA |
| GSE178166 |
The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration |
|
| Relations |
| BioSample |
SAMN19697465 |
| SRA |
SRX11141272 |
