Total RNA were extracted using Trizol. The RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Label
Cy3
Label protocol
Double-strand cDNA (ds-cDNA) was synthesized from total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). One μg ds-cDNA was incubated for 10 min at 98°C with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled ds-cDNA was purified by isopropanol / ethanol precipitation.
Hybridization protocol
Microarrays were hybridized at 42°C during 16 to 20h with 4 μg of Cy3 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA). After being washed in an ozone-free environment, the slides were scanned using the Axon GenePix 4000B microarray scanner.
Scan protocol
Slides were scanned at 5 μm/pixel resolution using an Axon GenePix 4000B scanner (Molecular Devices Corporation) piloted by GenePix Pro 6.0 software (Axon).Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis.
Description
Gene expression of B7-H3 CAR-T
Data processing
Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization.All gene level files were imported into Agilent GeneSpring GX software (version 11.5.1) for further analysis. Differentially expressed genes were identified through Fold Change filtering.
