Title: Fluidics Station Protocol. Description:
A first scanning was performed after the first SAPE staining. After performing secondary stainings a second scan was done, according to the standard Affymetrix scan procedures.
(Parameters: Scanning hardware = GeneChip Scanner 3000 [Affymetrix], Scanning software = Scanning software)
- equilibrate probe arrays to room temperature immediately before use
- wet the array by filling it with the appropriate volume of 1X Hybridization buffer
- Incubate at 45C for 10 minutes with rotation at 60rpm.
- Remove the buffer and replace with appropriate volume of clarified hybridization cocktail
- Place the probe array in the rotisserie
- hybridize for 16 hours at 45C, with a rotation of 60rpm.
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 10, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume unit = Nano litre, temperature = 45)
Biotin-labelled cRNA targets were obtained from 3 ug of total RNA. cDNA synthesis was performed with SuperScript cDNA Synthesis Kit (Invitrogen), and biotin-labelled cRNA was obtained using Megascript In vitro Transcription System (Ambion) including Bio-11-UTP and Bio-11-CTP in the reaction.
(Parameters: Amount of nucleic acid labeled = 3, Label used = OTHER: biotin, Amplification = none, Mass unit = Micro gram)
Total RNA was extracted using the RNeasy extraction kit (Qiagen)according to the manufacturer's instructions
(Parameters: Extracted product = total_RNA, Amplification = none)
siRNA duplexes were prepared by annealing pairs of 21-ribonucleotides (Dharmacon Research) and transfected using OLIGOFECTAMINE (Invitrogen) according to the manufacturer’s instructions.
HeLa cells were treated for 36 hours with siRNA oligos against HectH9 or Lamin A/C
Cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 2mM glutamine and 100U/ml streptomycin
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = DEMEM)