Primescript 2

The genomic DNA and transcripts in the petals of the genome-edited lines and the WT control underwent an Illumina NGS analysis. Genomic DNA was isolated from the leaves of transgenic and WT gentian plants with the GeneElute Genome DNA Isolation system (Sigma-Aldrich, St Louis, MO, USA). Total RNA was isolated from the petals of transgenic gentian plants with the RNeasy Plant Mini kit (Qiagen, GmbH, Hilden, Germany) and treated with DNase I (TaKaRa) according to the manufacturer’s instructions. The extracted RNA samples were reverse transcribed with PrimeScript II (TaKaRa). The subsequent library preparation, PCR amplification, amplification check, and library quantification check were performed as previously described^{18 (link)}. The primer positions and target sites are indicated in Fig. 2b. Additional details regarding the primers (e.g., index) are listed in Supplementary Table S1. A bioinformatics analysis was performed as previously described^{18 (link)}. Briefly, the resulting raw sequence reads were pre-processed with the FASTX toolkit, and the read pairs were merged into a single contiguous sequence (fragment) with a fastq-join script^{28 (link)}. The unique fragments were then counted.

The expression results of twelve selected unigenes were chosen for confirmation with qRT-PCR assay. The total RNA content was extracted from individual samples and the first strand cDNA templates were prepared using the PrimeScript II reverse transcriptase system (TaKaRa). Primers for each unigene were designed using Primer3 software. The experiment was performed using the qRT PCR system ABI7500 Real Time System (Applied Biosystems) using SYBR Green I (Roche). Each reaction was carried out in a total of 20 μL volume following the reaction conditions: 94 °C 10 min; 94 °C 15 s, 60 °C 10 s and 72 °C 25 s for 40 cycles. Three independent biological replicates were used for each sample and the actin gene was kept as an internal reference gene. The relative expression of genes was calculated by using the 2 − ^△△Ct method.

Total RNA was isolated from the cells by using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were determined by standard spectrophotometric methods using a NanoDrop ND-2000 (NanoDrop Technologies). cDNA was synthesized from 1 µg total RNA using the PrimeScript II reverse transcriptase (Takara Bio Inc.). Expression of p-EMT, EMT and epithelial differentiation-relatedgenes were measured using TB Green Premix Ex Taq II (Takara Bio Inc.) with a LightCycler 96 system (Roche). The primers used in this study are shown in Supplementary Table S1. Relative mRNA expression of each transcript was normalized to the endogenous control GAPDH mRNA and were calculated using the comparative cycle threshold method.
Heatmap was generated by R package pheatmap. Expression values were centered and scaled in the row direction, each gene.

Extraction and purification of total RNA were performed using RNAiso Plus (Takara, Japan). Then cDNA was synthesized with random nonamer and oligo dT(18) using PrimeScript II reverse transcriptase (Takara). qPCR was conducted using GoTaq qPCR mix and run on a CFX96 Touch Deep Well (Bio-Rad, Hercules, CA, USA). Relative quantification was done by standard curve method, and the expression levels of target genes were normalized against that of the reference gene, ornithine decarboxylase antizyme (OAZ1).

At day 3 post surgery, tissue was recovered from some animals and lysed using Trizol with mechanical homogenization to extract RNA. 1 μg RNA was extracted, treated with DNAse, and transcribed to cDNA using PrimeScriptII (Takara). Per 25 μL reaction, 1 μL of transcript was added to 12.5 μL ddPCR Supermix for probes (Biorad), 2.5 μL Taqman probe (Life Technologies), and 9 μL water. Droplets were generated by adding the 25 to 70 μL Droplet oil (Biorad) using a QX200 droplet generator (Biorad). Amplification was carried out using standard Taqman protocols using a PCR thermal cycler (Biorad). Samples were analyzed using a QX100 droplet reader (Biorad). Thresholding was manually performed, based on negative and positive control results when automatic thresholding of values was not possible.

The isolation of total RNA from HCT-116 cells was done with the RNA Isolater Total RNA Extraction Reagent (CAT#R401-01, Vazyme). The binding site of miR-450-5p at wild-type LINC00641 (LINC00641 WT) was amplified from total RNA using PrimeScript II (CAT#6210A, Takara, Tokyo-to, Japan) and cloned in the pmirGLO vector (CAT#E1330, Promega, Madison, United States), downstream of the firefly luciferase reporter gene. The wild-type 3′UTR of the GOLPH3 gene (GOLPH3 3′UTR-WT) was PCR amplified from the genomic DNA of HCT-116. The GOLPH3 3′UTR fragment was also cloned into the pmirGLO vector, downstream of the firefly luciferase reporter gene. The generation of the mutant versions of pmirGLO-LINC00641 WT and pmirGLO-GOLPH3 3′UTR (pmirGLO-LINC00641 MUT and pmirGLO-GOLPH3 3′UTR MUT) was done with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, United States) as documented by the manufacturer Lentiviral plasmid pLKO.1-TRC-MCS-T2A-puro (Antihela, Xiamen, Fujian, China) was utilized for knockdown of LINC00641 plasmid. Notably, hsa-miR-450b-5p mimic and mimic NC were supplied by Ribobio (Guangzhou, China). Thereafter, the cDNA of GOLPH3 was reverse transcribed from total RNA using PrimeScript II and inserted into the overexpression vector pCDH-EF1a-MCS-T2A-bsd (Antihela). Table S2 provides the primer sequences utilized for plasmid construction.

Total RNA extracts of human culture Jurkat cell line (RCB3052, RIKEN BioResource Research Center) were a kind gift of Ryotah Uehara (Hokkaido University) and total RNA extracts of adult Drosophila were prepared by using FastGeneTM kit (Nippon Genetics). cDNA libraries were synthesized by using 100 ng total RNA and with random 6-mer primer with PrimeScript II (Takara Bio Inc) by following the manufacturer protocol.