Genejet endo free plasmid maxiprep kit

DNA coding NMU was obtained by reverse transcription of total RNA isolated from SW480 cells, by the use of SuperScript One-Step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher Scientific) with primers: 5’-AGCTAAGCTTGCCGAGATGCTGCGAACAGAGAG-3’ and 5’-GGTCAGCAGGGTTCATTTAACGCGGATCCAATAGC-3’. PCR product was cloned into pJET1.2/blunt with the Clone JET PCR Cloning Kit (Thermo Fisher Scientific) using HindIII and BamHI restriction sites and subsequently to the pcDNA 3.1(+) (Thermo Fisher Scientific). The obtained pcDNA-NMU was propagated in Escherichia coli TOP10, purified with GeneJET Endo-Free Plasmid Maxiprep Kit (Thermo Fisher Scientific), and sequenced. Plasmids containing the NMU sequence were transfected into HT29 cells using Amaxa Cell Line Nucleofector Kit R and Amaxa 4D nucleofector X Unit (Basel, Switzerland). Caco-2 was chemically transfected using the Xfect ™ RNA Transfection Reagent (Takara Bio Inc., Kusatsu, Japan). Subsequently, the cells were cultured in medium supplemented with Hygromycin B (Thermo Fisher Scientific) 400 µg/ml for HT29 and 250 µg/ml for Caco-2. The selection medium was refreshed every 48 h. After 4 weeks in culture, well-separated colonies were isolated. NMU expression was verified through Western blot analysis.

CRISPR/Cas9 plasmids were generated as described in the Genome-Scale CRISPR Knock-out (GeCKO) protocol (Supplementary Table 2) [20 (link),24 (link)]. The plasmid pLentiCRISPR.v2 (Addgene #52961), which also contains a Puromycin-resistance cassette, was BsmBI digested to remove the filler fragment (1.89kb) and gel purified. Complementary DNA oligonucleotides containing selected CRISPR/Cas9 target sequences (Supplementary Table 3) were phosphorylated, annealed and ligated into the vector. Subsequently, plasmids were transformed into E. coli Stbl3 by heat-shock and colonies were screened by PCR (Supplemental Procedures). Positive clones were propagated followed by plasmid purification using the EndoFree Plasmid MaxiKit (Qiagen) or the GeneJET Endo-free Plasmid Maxiprep Kit (Thermo Scientific) according to the supplier´s protocol. Next, plasmids were confirmed by analytical digestions (BamHI: linearization of plasmid yielding one 12.98kb fragment; BamHI/EcoRV: yielding one 10.66kb and one 2.32kb fragment) and sequencing. Final plasmids are designated as pLentiCRISPR.v2-GFP, pLentiCRISPR.v2-SPRY1#1 and pLentiCRISPR.v2-SPRY1#2. pLentiCRISPR.v2-GFP and pLentiCRISPR.v2-SPRY1#1 were already employed in our previous study [13 ]. shRNA-encoding vectors required for SPRY1 gene knockdown were previously described [13 ]. A non-targeting shRNA was employed for comparison [10 (link)].