Fibroblasts were transduced with the retrovirus mixtures as indicated and DMSO, diclofenac, EP4 antagonist, PGE2, TGFb inhibitor or Wnt inhibitor was added simultaneously. The medium was replaced with FBS medium, which contained DMEM (high glucose) with L-glutamate and phenol red (Wako, Tokyo, Japan, 044-29765); Medium199 with Earle’s Salts, L-glutamate and 22 g/L sodium bicarbonate (Gibco, 11150-059); and 10% Hyclone Characterized FBS (Thermo Scientific, SV30014.03), after 24 h of infection and changed every 3 days.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from GHMT-, GHMT/diclofenac-, GHMT/EP4 antagonist-, GHMT/diclofenac/PGE2-, GHMT/TGFb inhibitor- or GHMT/Wnt inhibitor-induced TTFs using the ReliaPrep RNA Cell Miniprep System (Promega, Z6012) quantified using a NanoDrop-2000c spectrophotometer. Quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
biotin
Label protocol
Biotinylated ss-cDNA were prepared according to the standard Affymetrix protocol from 100ng total RNA (GeneChip WT PLUS Reagent Kit User Manual, Affymetrix).Biotinylated ss-cDNA yield were checked with the NanoDrop ND-2000c Spectrophotometer.
Hybridization protocol
Fragmented and labeled ss-cDNA were hybridized for 16 hr at 45°C on GeneChip Clariom S Array. GeneChips were washed and stained in the GeneChip Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 7G.
Description
SAMPLE 5
Data processing
Raw data were processed with Affymetrix Expression Console software version 1.4 for normalization.