Genechip fluidics station 450

The required reagents Agilent RNA 6000 Nano Kit, GeneChip 3’IVT Express Kit, GeneChip Hybridization Wash and Stain Kit, Trizol Kit, and QIAGEN RNeasy Total RNA Isolation kit were provided by Gekkai Gene Technology Co., Ltd. The main instruments used in the study included the Thremo Nanodrop 2000, Agilent 2100, GeneChip Hybridization Oven 645, GeneChip Fluidics Station 450, and GeneChip Scanner 3000.

RNA was isolated from human cell culture using Isol-RNA lysis procedure (Trizol, Thermo Fisher Scientific). RNA was DNaseI (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega). Quantitative RT–PCR was performed in triplicate with SYBR select (Thermo Fisher Scientific) using Rotor-Gene 3000 (Qiagen) and normalised to GAPDH/ACTB. We performed RNA microarrays (Clariom S human) according to manufacturer's protocol (P/N 703174 Rev. 2) with 200 ng of total RNA. Reagents/equipment were GeneChip WT PLUS Reagent Kit, P/N: 902280; GeneChip Hybridization, Wash, and Stain Kit P/N 900720, GeneChip Scanner, GeneChip Fluidics Station 450, GeneChip Hybridization Oven 640, Bioanalyzer 2100 (Agilent), and RNA600 NanoKit (Agilent).

RNA was isolated from human cell culture or mouse primary tissues (homogenized using Bioruptor, Diagenode) using Isol-RNA lysis procedure (Trizol, Thermo Fisher Scientific). RNA was DNase (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega). Quantitative RT–PCR was performed in triplicate with SYBR select (Thermo Fisher Scientific) using Rotor-Gene 3000 (Qiagen). For primers see Supplementary Table S2. RNA microarrays (MoGene-2_0-st, Thermo Fisher Scientific) were performed according to manufacturer’s protocol (P/N 703174 Rev. 2) with 200 ng of total RNA. Reagents/equipments were GeneChip® WT PLUS Reagent Kit, P/N: 902280; GeneChip® Hybridization, Wash, and Stain Kit P/N 900720, GeneChip Scanner, GeneChip Fluidics Station 450, GeneChip Hybridization Oven 640, Bioanalyzer 2100 (Agilent) and RNA600 NanoKit (Agilent). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk) under accession number E-MTAB-8296.