Cells were grown at a dense or sparse cell density as described in the growth protocol.
Growth protocol
The FAK-Knockout cell line is described here: http://www.nature.com/nature/journal/v377/n6549/pdf/377539a0.pdf (Ilic D et al., Nature, 1995) The FAK-Rescue cell line is described here: http://jcs.biologists.org/content/112/16/2677.short (Sieg DJ et al., JCS, 1999; clone DA2) FAK Rescue were cultivated and seeded for experiments in DMEM + Glutamax + 10% FBS + 135ug/ml HygromycinB + non essential amino acids. FAK KO were cultivated and seeded for experimentsin DMEM + Glutamax + 10% FBS (without any further antibiotics). Cells were cultivated in 15cm dishes with 30ml medium ot the respective medium for at least 2 days before the initial seeding for a microarray. Cells were split at at least once within 2 days to keep a constant confluency ~50% for cultivating. For seeding up to three 15cm dishes containing 90% confluent cells were pooled. Cells were harvested with 5ml Trypsin, followed by resuspension in 30ml medium , centrifugation at 250g for 5min, removing of s/n, resuspension in medium. Then cells aggregates were dissolved by pipetting up and down >20 times while pressing the pipet to the bottom of a plate. For dense condition, 8.2 x10^6 cells were seeded into a 10cm dish with 10ml medium. For sparse condition 0.41 x10^6 cells were seeded into a 10cm dish with 10ml medium. Cells were the grown at 37deg for 24+/-1h until being harvested. The position of the plates within the incubator would change for individual samples among replicate days to reduce positional bias. The seeding/harvesting was done on three different experimental days.
Extracted molecule
total RNA
Extraction protocol
Cells were harvested by trypsinization and frozen at -80deg. RNA preparation was done on the same day for all samples. Sample preparation was done with the Qiagen RNeasy Mini Kit including the optional on column DNAse treatment according to the manufacturer's manual, except: used 0.8mm syringe for homogenization instead of recommended 0.9mm syringe.
Label
Cy3
Label protocol
The quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies,Delaware, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a RNA Integrity Number (RIN) higher than 8 were further processed. Total RNA samples (100ng) were reverse-transcribed into double-stranded cDNA in presence of RNA poly-A controls, RNA Spike-In Kit, One-Color (Agilent p/n 5188-5282). The double-stranded cDNA were in vitro transcribed in presence of Cy3-labelled nucleotides using a Low Input Quick Amp Labeling Kit, one-color (Agilent P/N 5190-2305). The Cy3- cRNA was purified using a RNeasy mini kit, Qiagen (p/n. 74104 or 74106) and its quality and quantity was determined using NanoDrop ND 1000 and Bioanalyzer 2100. Only cRNA samples with a total cRNA yield higher than 2 μg and a dye incorporation rate between 8 pmol/ μg and 20 pmol/ μg were considered for hybridization
Hybridization protocol
Cy3-labeled cRNA samples (1.65 μg) were mixed with a Agilent Blocking Solution, subsequently randomly fragmented to 100-200 bp at 65°C with Fragmentation Buffer, and resuspended in Hybridization Buffer using a Gene Expression Hybridization Kit (Agilent p/n 5188-5242). Target cRNA Samples (100μl) were hybridized to Whole Mouse Genome 4x44k OligoMicroarrays (Agilent G4122F) for 17h at 65°C. Arrays were then washed using Agilent GE Wash Buffers 1 and 2 (Agilent p/n 5188-5326), according to the manufacturer instructions (One-Color Microarray-Based Gene Expression Analysis Manual, www.agilent.com)
Scan protocol
An Agilent Microarray Scanner (Agilent p/n G2565BA) was used to measure the fluorescent intensity emitted by the labeled target.
Description
FAK Rescue, dense, Experimental Day July12th
Data processing
Raw data processing was performed using the Agilent Scan Control and the Agilent Feature Extraction Software Version 10. Quality control measures were considered before performing the statistical analysis. These included, inspection of the array hybridization pattern (absence of scratches, bubbles, areas of non hybridization), proper grid alignment, performance of the spike in controls (linear dynamic range between 5 orders of magnitude) and number of green feature non uniformity outliers (below 100 for all samples). Matrix table values are intensities obtained by Agilent feature extraction software, followed by geometric mean normalization. 'null' values indicate measurements below detection backround. To be above background, measurments had to fullfill the default Agilent output gIsWellAboveBG and additional threshold of 25 fluorescence units. Both criteria had to be satisied in at least 2 out of 3 biological replicas for that gene/condition.
