Total RNA was extracted with Trizol reagent (ThermoFisher Scientific) according to the manufacturer's instructions.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
0.6 ug of Cy3-labelled cRNA (specific activity >25.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Fragmentation Buffer (Agilent) and 2x Blocking Agent (Agilent) following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x GE Hybridization Buffer HI-RPM(Agilent) was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE microarry 8x60K Ver. 3.0 (G4851C, Agilent Technologies) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2539A) using one color scan setting for 1x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Description
Gene expression two days after plating.
Data processing
The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters (protocol AgilentG3_GX_1Color and Grid: 072363_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities.
Knock down of MTHFD2 of lung adenocarcinoma cell line H322
Data table header descriptions
ID_REF
VALUE
Data were normalized and filtered with GeneSpring software 13 (Agilent Technologies). In brief, raw data were normalized with their 75 percentile values. With respect to the filtering, we exploited three filters. In the first filter, when a value of a probe was lower than 20 percentile value in any of the six samples, the probe was excluded. In the second filter, when the flag of a probe was compromised in any of the samples, the probe was excluded. In the last filter, when the value (log scale) of CV was over 50 percent in both conditions, the probe was excluded. After filtering, control probes were excluded, and the log scale values were transformed into normal scale.
