cell type: Human retinal pigment epithelial (hRPE) lentivirus: shFOXD1
Treatment protocol
shRNAmir lentiviral vectors were obtained from Thermo Scientific. High-titer lentiviral particles for each plasmid were used to transduce hRPE cells (ScienCell, cat. #6540). Twenty-four hours after infection, epithelial cell medium was replaced and selection with 1 μg/ml puromycin (Life Technologies, cat. #A1113803) was carried out. Puromycin-resistant cells were harvested for future analysis five days after transduction. Additional details are provided in the Extended Experimental Procedures.
Growth protocol
Human retinal pigment epithelial (hRPE) cells used for knockdown experiments were purchased from ScienCell (ScienCell, cat. #6540). hRPE cells were maintained in epithelial cell medium (EpiCM) (ScienCell, cat. #4101) supplemented with 2% fetal bovine serum (ScienCell, cat. #0010), 1x epithelial cell growth supplement (EpiCGS) (ScienCell, cat. #4152), and 1x penicillin/streptomycin solution (ScienCell, cat. #0503). Human foreskin fibroblasts (HFF) were purchased from GlobalStem (GlobalStem, cat. #GSC-3002) and maintained in DMEM (Life Technologies, cat. #11965-092) supplemented with 15% of Tet System Approved fetal bovine serum (Clontech, cat. #631101), 2mM L-Glutamine (Life Technologies, cat. #25030-081) and 100 U/ml penicillin-streptomycin (Life Technologies, cat. #15140-163).
Extracted molecule
total RNA
Extraction protocol
Total RNA was harvested and used for library preparation. For each transcription factor, total RNA was harvested from two different lines, each harboring a different shRNAmir construct. 100 ng of total RNA was used to prepare biotinylated cRNA (cRNA) using the 3’ IVT Express Kit (Affymetrix, cat. #901228), following the manufacturer’s suggested protocol.
Label
biotin
Label protocol
Biotinylated RNA was prepared according to the standard Affymetrix protocol from 100 ng total RNA (GeneChip 3' IVT Express Kit User Manual, 2009, Affymetrix).
Hybridization protocol
Following fragmentation, 12.5 ug of aRNA were hybridized at 45C for 16 hr at 60RPM on GeneChip Arrays (PrimeView). GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the manurfacturer's instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 and images were extracted with Affymetrix GeneChip Expression Console. A CDF containing the ERCC probes was provided by Affymetrix.
Description
RPE sh KD FOXD1 1
Data processing
A CDF provided by Affymetrix, which contained the ERCC probes (PrimeView_withERCC_binary.cdf) was used instead of the standard PrimeView cdf. The data were analyzed with Bioconductor using the MAS5 normalization method. Then, these data were renormalized to the ribosomal genes. See manuscript for additional details.
