We performed retroviral gene transduction for cells expressing short hairpin (sh) RNAs and lentiviral induction of Prex2a.
Growth protocol
JHUEM-14 were cultured in DMEM/ Ham F12 (Sigma) + 20% FBS (EQUITECH-BIO) + MEM NEAA (gibco). OMC-2 were cultured in Ham’s F-12 (Sigma) + 20% FBS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy Mini(QIAGEN).
Label
Cy3
Label protocol
According to the manufacturer’s protocol (One-Color Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling, Version 6.5, May 2010).
Hybridization protocol
According to the manufacturer’s protocol (One-Color Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling, Version 6.5, May 2010).
Scan protocol
According to the manufacturer’s protocol (One-Color Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling, Version 6.5, May 2010, Agilent C Scanner Settings).
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 072363_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities. The data set from the scanned microarrays were analyzed with GeneSpring software 13.1 (Agilent). The parameter settings for importing the data into GeneSpring were as follows: Threshold, 1.0; Logbase, 2; Technology, Agilent.SingleColor.72363; Normalization, Shift to 75 percentile; Baseline Transformation, None. Signal intensity after normalization by GeneSpring GX11.5 software (Agilent). [Threshold: 1.0, Logbase: 2, Normalization: Shift to 75 percentile, Baseline Transformation: None]
