Simplyrna tissue kit

Genomic DNA (gDNA) and total RNA were extracted from 25-30mg fresh frozen tumor tissue using the Tissue DNA Purification Kit and Simply RNA Tissue Kit respectively, and run on the Maxwell 16 instrument according to manufacturer’s protocol (Promega, Madison, WI, USA). Sample concentration and purity was measured with DS-11 Spectrophotometer (De Novix, http://www.denovix.com) and QuBit fluorometer (Thermo Fisher Scientific). The integrity of total RNA was assessed using a Bioanalyzer (Agilent Technologies, http://www.agilent.se), and RNA Integrity Numbers (RIN) from the six RNA samples were within the range of 6.7–8.6. A total amount of 750 ng total RNA was reverse transcribed into cDNA using SuperScript VILO cDNA Synthesis Kit according to manufacturer’s protocol (Thermo Fisher Scientific).

Each RNA was extracted according to the manufacturer’s instructions. The total mRNA from GC cells was extracted using a simplyRNA Tissue Kit (Promega Corporation, Madison, WI). In addition, mRNA extraction of tumour tissues removed from mice was performed using a RNeasy Mini (QIAGEN, Venlo, Netherlands). cDNA was synthesised using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) and a PCR Thermal Cycle Dice (Takara, Shiga, Japan). RT-PCR was performed using power up SYBR green Master mix (Thermo Fisher Scientific). Cycling conditions were as follows: one cycle each at 50 °C for 2 min and 95 °C for 2 min, and 45 cycles each at 95 °C for 15 sec and 60 °C for 1 min. RT-PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Data were normalised to β-actin. The breakdown of the specific primers is shown in Supplementary Table 1.

Gene-expression analyses were conducted on 34 HTE, 20 HTI, 22 MS, 7 MS + HT, and 56 HC. Total RNA was extracted using the automated Maxwell^® Rapid Sample Concentrator (RSC) Instrument, with Maxwell^® 16 LEV simplyRNA Blood Kit (Promega) for whole blood and simplyRNA Tissue kit (Promega) for PBMCs, following the manufacturer’s instructions. Total RNA was reverse-transcribed at a final concentration of 20 ng/µL using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Gene-expression analysis was performed by real-time PCR using TaqMan^® gene-expression products (Thermo Fisher Scientific). Expression levels of target genes were calculated by the normalized comparative cycle threshold (Ct) method (2^−ΔΔCt), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene and the Universal Human Reference RNA (Stratagene, Santa Clara, CA, USA) as calibrator.

RNA isolation was performed using the simplyRNA tissue kit (cat# AS1340, Promega Corporation), following manufacturer’s instructions. Briefly, tissue pieces were cut on dry ice with a sterile scalpel blade and placed in a Precellys lysing kit tube (cat# P000912-LYSKO-A, Bertin Corp, Montigny-le Bretonneux, France). Sample homogenization buffer containing 0.02% thioglycerol was added, and the tube was placed into a precooled Precellys device. The Precellys machine was then programmed to rupture hard tissue for 2 min. After the tissue was completely disintegrated, 200 µL of lysis buffer was added to the cell suspension and then transferred into a cartridge of Maxwell simplyRNA tissue kit (cat# AS1340, Promega Corporation), and 5 µL of DNase 1 was added to the appropriate well of the cartridge. RNA isolation was done following the installed kit-specific protocol and eluted in 60 µL of nuclease-free water. Quantification was carried out using a Quantus fluorometer (Promega), and samples were stored at −80 °C until analyses.

Total RNA was extracted using simplyRNA Tissue Kit (Promega). For RT-PCR analysis, cDNA was synthesized with the SuperScript™ VILO™ Master Mix (Thermo). RT-qPCR (Reverse Transcription Quantitative real-time PCR) was performed by using FastStart Universal SYBR Green Master (Roche). The expression of each gene was calibrated to Gapdh gene expression as a housekeeping gene. For RNA-seq studies, mRNA was isolated from total RNA by using NEBNext Poly(A) mRNA magnetic isolation module (NEB), and sequencing libraries were generated according to the NEBNext Ultra II RNA library prep kit for illumina (NEB) protocol. RT-qPCR primers are listed in Supplementary Table 1.