Total RNA from HCT116 cells treated with 50 μM curcumin for 24 h was extracted using Trizol reagent (Invitrogen). Amplification of the 5′-end of hST6GalNAc I was conducted with gene-specific primers (Table 1 ) and the GeneRacer kit (Invitrogen, United States) according to the manufacturer’s instructions, as previously described (Lee et al., 2018b (link)). The amplified product was analyzed on a 1% (w/v) agarose gel and purified using GeneAll Expin Gel SV kit (GeneAll biotechnology, Korea). The purified PCR product was subcloned into pGEM-T Easy vector (Promega, United States) and sequenced by Macrogen Company (Seoul, South Korea).
Expin gel sv kit
Manufactured by GeneAll
The Expin™ Gel SV kit is a lab equipment product designed for the extraction and purification of DNA from agarose gel slices. It provides a simple and efficient method to recover DNA fragments from agarose gels.
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Lab products found in correlation
Exprep plasmid sv kit, by GeneAll (2 mentions)
Generacer kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Ptg19 t pcr cloning vector, by Vivantis Technologies (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Exgene cell sv kit, by GeneAll (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
T vector, by Promega (1 mentions)
T7 rna polymerase, by New England Biolabs (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
7 protocols using expin gel sv kit
1
Amplification and Sequencing of hST6GalNAc I
2
Carob Moth Heat Shock Gene Sequencing
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Because the heat shock gene sequences in carob moth were unknown, primers were designed using alignments of gene sequences from other insects including Chilo suppressalis, Bombyx mori, Pieris brassicae, Spodoptera exigua, and Helicoverpa armigera. Degenerate primers were used to amplify ~550 bp gene fragments of the carob moth hsp70 and hsp90 genes, listed in Table 1 .
The amplification products were resolved by gel electrophoresis, bands were excised from the gel, and a Gene All Expin Gel SV kit (GeneAll) was used to isolate the DNA. The PCR products were ligated into a pTG19-T PCR Cloning Vector (Vivantis Technologies Sdn Bhd) using T4 DNA Ligase. The plasmids were used to transform E. coli DH5a cells using heat shock. Colonies with inserts were screened with blue/white X-gal selection under standard ampicillin conditions. Plasmid DNA was extracted from recombinant bacterial cells using the alkaline lysis method described by Sambrook and Russell [37 ], and the DNA was sequenced from multiple [5 (link)–10 ] different independent bacterial colonies by Bioneer (Daejeon, Korea).
The amplification products were resolved by gel electrophoresis, bands were excised from the gel, and a Gene All Expin Gel SV kit (GeneAll) was used to isolate the DNA. The PCR products were ligated into a pTG19-T PCR Cloning Vector (Vivantis Technologies Sdn Bhd) using T4 DNA Ligase. The plasmids were used to transform E. coli DH5a cells using heat shock. Colonies with inserts were screened with blue/white X-gal selection under standard ampicillin conditions. Plasmid DNA was extracted from recombinant bacterial cells using the alkaline lysis method described by Sambrook and Russell [37 ], and the DNA was sequenced from multiple [5 (link)–10 ] different independent bacterial colonies by Bioneer (Daejeon, Korea).
3
Plasmid Construction and Purification for Bacterial Studies
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The bacterial strains and plasmids used are listed in Supplementary Data 1 . Methods for plasmid construction are elaborated in Supplementary Note 3 . The primers were synthesized by Cosmogenetech (Seoul, Korea) and are listed in Supplementary Data 2 . Plasmid DNA was isolated using an ExprepTM Plasmid SV kit from GeneAll (Seoul, Korea). DNA fragments amplified by PCR were purified using an ExpinTM Gel SV kit (GeneAll). Q5 polymerase and restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The reagents for cell cultures were purchased from BD Bioscience (Sparks, MD, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA) as listed in Supplementary Data 2 , unless otherwise indicated.
4
Detection of O. tsutsugamushi by Nested PCR
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To confirm the presence of O. tsutsugamushi, a nested PCR targeting the 56-kDa gene of O. tsutsugamushi was performed. Primers 34 (forward, 5'-TCA AGC TTA TTG CTA GTG CAA . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint this version posted August 13, 2020. ; https://doi.org/10.1101/2020.08.13.249219 doi: bioRxiv preprint TGT CTGC-3'; the 56-kDa gene based on the Gilliam strain) and 55 (5'-AGG GAT CCC TGC TGC TGT GCT TGC TGCG-3') were used in the first PCR. Nested PCR primers 10 (5'-GAT CAA GCT TCC TCA GCC TAC TAT AAT GCC-3') and 11 (5'-CTA GGG ATC CCG ACA GAT GCA CTA TTA GGC-3') were used in the second PCR amplification to generate a 483bp fragment. Nested PCR was performed as described by Lee et al. [8] . The amplified PCR products were confirmed by agarose gel electrophoresis and purified from the agarose gel using the QIAquick gel extraction kit (QIAGEN) and the Expin TM Gel SV kit (GeneAll, Seoul, Korea). Each PCR product was analyzed and confirmed by sequencing.
The copyright holder for this preprint this version posted August 13, 2020. ; https://doi.org/10.1101/2020.08.13.249219 doi: bioRxiv preprint TGT CTGC-3'; the 56-kDa gene based on the Gilliam strain) and 55 (5'-AGG GAT CCC TGC TGC TGT GCT TGC TGCG-3') were used in the first PCR. Nested PCR primers 10 (5'-GAT CAA GCT TCC TCA GCC TAC TAT AAT GCC-3') and 11 (5'-CTA GGG ATC CCG ACA GAT GCA CTA TTA GGC-3') were used in the second PCR amplification to generate a 483bp fragment. Nested PCR was performed as described by Lee et al. [8] . The amplified PCR products were confirmed by agarose gel electrophoresis and purified from the agarose gel using the QIAquick gel extraction kit (QIAGEN) and the Expin TM Gel SV kit (GeneAll, Seoul, Korea). Each PCR product was analyzed and confirmed by sequencing.
5
Tryptophan Production in Bacterial Strains
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The bacterial strains and plasmids used in this study are listed in Table 3 . Mach-T1R was used as a host for all plasmid construction. The EPW used for tryptophan production is a strain in which the selectable marker of ATCC31743 has been replaced with a kanamycin resistance gene. Plasmid DNA and genomic DNA were isolated using Exprep™ Plasmid SV kit (GeneAll Biotechnology, Seoul, Korea) and ExgeneTM Cell SV kit (GeneAll Biotechnology), respectively. DNA fragments were purified using the Expin™ Gel SV kit (GeneAll Biotechnology). Q5 polymerase and NEBuilder® HiFi DNA Assembly Master Mix were purchased from New England Biolabs (Ipswich, MA, USA). Luria-Bertani (LB) broth and agar used for the cloning process of plasmids, and yeast extract, were obtained from BD Biosciences (Sparks, MD, USA). Other chemicals were attained from Sigma–Aldrich (St. Louis, MO, USA). The oligonucleotides were synthesized by Cosmogenetech (Seoul, Korea) (Table 1 ).
6
Single-molecule FRET and In vitro DNA Cleavage Assay
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For single-molecule FRET assay, all oligo nucleotides including DNA and RNA are purchased from Integrated DNA Technologies. NTS of the dsDNA has two types of modifications: 5′ end biotinylation for surface immobilization and internal amino modification for dye labeling. For gRNA, 5′ end Cy5 labeled crRNA and AltR-tracrRNA were purchased from IDT. Detailed information for DNA or RNA sequences with various modifications are listed in Supplementary Table S1 .
For in vitro DNA cleavage assay, the EMX1 target sequence was obtained from human genomic DNA and Gibson assembled into T-Vector (Promega). Based on the plasmid containing the EMX1 on-target sequence, the other plasmids containing the mismatched sequences were also generated by Gibson assembly. On- and Off-target DNAs were amplified by polymerase chain reaction (PCR) from the cloned plasmids. The PCR amplicons were analyzed on agarose gel, and then purified using Expin Gel SV kit (GeneAll, Seoul, Korea). Single-guide RNA targeting the EMX1 site was prepared by in vitro transcription. The RNA was transcribed by T7 RNA polymerase (New England Biolabs) and the product was purified with a RNeasy MinElute Cleanup Kit (QIAGEN).
For in vitro DNA cleavage assay, the EMX1 target sequence was obtained from human genomic DNA and Gibson assembled into T-Vector (Promega). Based on the plasmid containing the EMX1 on-target sequence, the other plasmids containing the mismatched sequences were also generated by Gibson assembly. On- and Off-target DNAs were amplified by polymerase chain reaction (PCR) from the cloned plasmids. The PCR amplicons were analyzed on agarose gel, and then purified using Expin Gel SV kit (GeneAll, Seoul, Korea). Single-guide RNA targeting the EMX1 site was prepared by in vitro transcription. The RNA was transcribed by T7 RNA polymerase (New England Biolabs) and the product was purified with a RNeasy MinElute Cleanup Kit (QIAGEN).
7
Bisulfite Sequencing of miR-9 Promoters
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Genomic DNA was extracted from uncompressed, compressed CAFs, MDA-MB-231, and BT-474 using an Expin Gel SV Kit (GeneAll Biotechnology, Seoul, South Korea). For bisulfite conversion of unmethylated CpGs, 4 μg of gDNA was modified with sodium bisulfite using an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's manual. For methylation-specific qPCR analysis, methylated DNA-specific and unmethylated DNA-specific primer sets were designed by analyzing CpG islands in the 2 kb upstream of mir-9-1, -2, and -3 using MethPrimer,54 (link) as shown in Supplementary Figure S3 and Supplementary Table S2 . Relative methylation was calculated using the ΔΔCt method. The Ct values of methylated DNA were normalized to those of unmethylated DNA.
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