Day 1. Seeding 300000 per well (in 6-well plates) using complete media, Day 2: starvation for 24 hours, Day 3: treatment with 5nM metformin dissolved in the media 106. For the vehicle group, we just change the media
Growth protocol
Medium: medium 106 (Invitrogen) supplemented by 10% BCS
Extracted molecule
total RNA
Extraction protocol
QIAgen Rneasy mini kit
Label
Cy3
Label protocol
Purified total RNA was amplified using the Agilent Low-Input QuickAmp Kit (Agilent, Waldbronn, Germany).
Hybridization protocol
Hybridization was performed following the Agilent protocol at 65°C over-night in an in-situ hybridization oven with slides mounted in Agilent hybridization chambers. Slides were rotated with 2 rpm.
Scan protocol
Slides were scanned using the InnoScan 900 scanner (Innopsys, Carbonne France) at 2 µm/pixel resolution and analyzed with Mapix 6.5.0
Data processing
Raw data was further processed using R (http://www.R-project.org) and limma (Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. NAR 43(7), e47). Background correction was performed with the normexp model using negative control spots and and offset of 1. Spot intensities were quantile normalized between arrays.
