Rnase mini kit

Total RNA was isolated from tumor tissues of different groups using RNase Mini Kit (Qiagen, Valencia, CA, USA) and following kit instructions. Primer sequences were designed by using Beacon Designer software package (Bio-Rad). The sequences of primers were as follows: CXCL12 sense 5′-GCT ACA GAT GCC CAT GCC GAT-3′ and anti-sense 5′-AGC TTC GGG TCA ATG CAC ACT-3′, GAPDH sense 5′-TCA CCA CCA TGG AGA AGGC-3′ and anti-sense 5′-GCT AAG CAG TTG GTG GTG CA-3′, VEGFA sense 5′-CTG TGC AGG CTG CTG TAA CG-3′ and anti-sense 5′-GTT CCC GAA ACC CTG AGG AG-3′. All primers were synthesized by Invitrogen (Groningen, Netherlands). Real-time quantitative polymerase chain reaction (qPCR) for cDNA analysis was conducted at 60–95°C for 45 cycles on a Sequence Detection System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) by following the instructions given with the kit and using SYBR Green Reaction Master Mix (TaKaRa Biotechnology Co. Ltd., Dalian, China). For each sample, GAPDH served as the housekeeping gene. Fold-change expression was calculated from the threshold cycle values. For the calculation of relative changes, the gene expression measured in sham-irradiated tissues was taken as baseline value.

RNAs were isolated from DLD‐1 and ARPC2 knockout DLD‐1 cells using an RNase mini kit (Qiagen). Isolated RNAs were quantitated, and quality was measured in an agarose gel. For RNA‐seq, RNA libraries were generated with TruSeq RNA Sample Prep Kit v2 (Illumina), and size of the RNA library (250‐650 bp) was confirmed in 2% agarose gel. To analyze sequencing, samples that were prepared to 10 nmol/L were assayed using Hi‐Seq 2000 for 100 cycles and paired‐end sequencing (Illumina). Four RNA libraries were pooled in each lane for sequencing, and an average of approximately 11 Gb was obtained for each sample. After mapping using a reference database, gene set analysis and pathway analysis were carried out through the RPKM normalization process and DEG selection.

Total RNA was prepared from raw crypts (freshly isolated crypts from mice) and cultured crypts using an RNase mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer's protocol. A total of 1 μg of RNA was reverse transcribed using an AccuPower RT PreMix kit (Bioneer, Seoul, South Korea). Real-time PCR was performed with FastStart Essential DNA Green Master Mix (Roche, Indianapolis, IN, United States). All reactions were performed in triplicate. mRNA expression was normalized to endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and expressed relative to ENR-derived cells or raw crypts. The primers sequences are listed in Table 1.