C1000

Three YWHAB sgRNAs were designed using online programs, E-CRISP [12 (link)] and CHOPCHOP [13 (link),14 (link),15 (link)], and subcloned into pCAG-eCas9-GFP-U6-gRNA vector (a gift from Jizhong Zou; Addgene plasmid # 79145) with an EGFP reporter as well as overhangs compatible for cloning into the Bbs1 site following digestion with Bbs1 restriction enzyme (Thermo Scientific, Waltham, MA, USA, ER1011). Complementary oligos of three YWHAB target sequences (Table 1) were heated at 37 °C for 30 min, 95 °C for 5 min, and annealed by decreasing the temperature 1 °C/min to 25 °C using a thermocycler (BioRad C1000; BioRad, Hercules, CA, USA). Next, the short double-stranded DNA fragments were ligated into the linearized Bbs1 site of the pCAG-eCas9-GFP-U6-gRNA vector. Ligation products were transformed into Escherichia coli (E. coli) NEB 5-alpha competent cells (New England Biolabs, Ipswich, MA, USA) and grown in LB (Luria–Bertani) medium containing 100 µg/mL ampicillin (Sigma–Aldrich, St. Louis, MO, USA) as a growth-selective marker. The cloned constructs were then purified using a ZR Plasmid Miniprep-Classic kit, sequenced (Sanger Sequencing), and analyzed using SnapGene software (from GSL Biotech; V6.0 snapgene.com, accessed on March 15, 2021; Boston, MA, USA) to confirm correct sgRNA insertion into the Bbs1 site.

After DNA extraction, the quality of all DNA samples was confirmed by PCR using the host gene; receptor tyrosine-protein kinase (erbB-2). The reference sequences of erbB2 for three species (sheep, goat, and cattle) were obtained from GenBank. The sequences were aligned and the primers were designed according to the conserved region of all species. A set of forward erbB2-F (5′-AGAACCTGCGAGTAATCC-3′) and reverse primers erbB2-R (5′-CCTTCTCTTCACTATACATACAC-3′) were used to amplify a 128-base pair (sheep and goat) and a 126-base pair (cattle) of the erbB2 region. The DNA fragments of the mentioned region were amplified in a total volume of 25 µl containing 12.5 µl of Premix (Ampliqon, Odense, Denmark), 1 µl MgCl2 (25 mM), 0.75 µl of each primers (25 pmol), 3 µl template DNA and 7 µl Double-distilled water (DDW) in automatic thermo cycler (Bio-Rad C1000; Bio-Rad, Hercules, CA, USA) under the following conditions: 93 °C for 3 min as initial denaturation followed by 35 cycle at 93 °C for 30 s as denaturation, 58 °C for 30 s as annealing, 72 °C for 30 s as extension, and final extension at 72 °C for 3 min. Samples with 3 µl of DDW instead of DNA was used as Blank. 5 µl of the PCR products were analyzed through electrophoresis in a 1.5% agarose gel (BioNeer, Daejeon, South Korea) in 1X TBE buffer at 90 V for 30 min and were visualized and photographed on transilluminator (UVITEC).

Total RNA was extracted from MSCs using the EasyBlue RNA isolation reagent (Intron Biotechnology, Sungnam, Korea). cDNAs were synthesized from 2 μg total RNA using the AccuPower cDNA synthesis kit (Bioneer, Daejeon, Korea). Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using the AccuPower PCR premix (Bioneer) in a thermal cycler (Bio-Rad C1000; Bio-Rad, Hercules, CA, USA). The amplified PCR products were electrophoresed on 1% agarose gels containing SybrSafe (Invitrogen, Carlsbad, CA, USA) and analyzed using a fluorescence image analyzer (LAS4000 mini; Fujifilm, Tokyo, Japan). PCR was performed with the following primers: IL-10 (forward 5′-ATCCAAGACAACACTACTAA-3′ and reverse 5′-TAA ATATCCTCAAAGTTCC-3′; product 587 bp) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; forward 5′-CCACTGGCGTCTTCACCAC-3′ and reverse 5′-CCTGCTTCACCACCTTCTTG-3′; 476 bp). To confirm the expression of ICOSL mRNA, quantitative RT-PCR (qPCR) was performed using the ICOSL TaqMan assay (Assay ID: Hs00323621_m1; Applied Biosystems, Waltham, MA, USA) and the TaqMan Universal PCR Master Mix (Applied Biosystems). The mRNA expression level was normalized to 18S rRNA gene expression (Hs03928985_g1) as an internal control. The qPCR was run on the StepOnePlus real-time PCR System (Applied Biosystems).

DNA from stool samples of CRC patients and health controls was extracted by E.Z.N.A stool DNA kit (Omega Bio-Tek, USA) according to the manufacture’s instructions. PCR for Fn genes was performed on a PCR Amplifier (BIO-RAD C1000, USA). The primers used in real-time PCR reaction were as follows: forward: 5′-ATA CCG GGA ATA AAG ACA-3′; reverse: 5′-TAC AAC CCA ATC CAT AAG T -3′.

ddPCR for analysis of expression level of the miRNAs in BAL samples was performed as described in our previous work [38 (link), 39 (link)]. Briefly, TaqMan™ reaction mix (Applied Biosystems) containing sample cDNA was partitioned into aqueous droplets in oil via the QX100 Droplet Generator (Bio-Rad, Pleasanton, CA, USA), and then transferred to a 96-well PCR plate. A two-step thermocycling protocol (95°C ×10min; 40 cycles of [94°C ×30s, 60°C ×60s], 98°C ×10 min) was undertaken in a Bio-Rad C1000 (Bio-Rad). The PCR plate was loaded on Droplet Reader (Bio-Rad), by which copy number of each miRNA per μl PCR reaction mixture was directly determined. Expression of a targeted miRNA in a given sample was calculated using the same equation as described above. However, instead of using Ct, we used copy number of gene in the equation: expression of a miRNA in a given sample = 2−Δcopy number of gene, where Δcopy number of gene = copy number of gene (targeted miRNA) – copy number of gene (U6).