RNA was linearly amplified by T7 RNA polymerase using two rounds of in-vitro transcription (MessageAmp-II kit Life Technologies) according to the manufacturer’s recommended protocol with some modifications. Cells were lysed by repeated heating to 70C and snap cooling to 4C and first strand synthesis was carried out at 42C for 2hrs with first strand buffer, dNTP mix, RNase inhibitor and ArrayScript enzyme. Second strand synthesis was done at 16C for 2hrs with second strand buffer, dNTP mix, T4 DNA polymerase and RNAseH. cDNA was purified using columns and first round IVT was performed at 37C for 14hrs to make aRNA. For the 2nd round of linear amplification column purified aRNA from first IVT underwent another first strand synthesis at 42C for 2hrs using second round primers, followed by RNaseH digestion at 37C for 30mins and another 2nd round second strand synthesis at 16C with a T7-RA5 primer. The resulting double stranded cDNA underwent a final second IVT step at 37C for 14hrs to make aRNA. These two rounds of linearly amplified aRNA products now carried the 3′-end of the polyA
