Wizard kit

Flash-frozen human I-NETs were obtained from the CHTN. Informed consent was obtained from CHTN and experiments were approved by the Institutional Review Boards of CHTN. Genomic DNA was prepared from 57 tumors using a Wizard kit from Promega. RNA was isolated from 42 tumors using an RNeasy kit from Qiagen. RNA was converted into cDNA using a reverse transcription reagents kit (Thermofisher), and CDK4 was quantitated using real-time RT-PCR, then normalized to the expression of β-actin. MicroRNA was prepared from 23 frozen tumors using a miRNeasy kit from Qiagen and converted into cDNA using Taqman advanced miRNA cDNA kit (Thermofisher). MIR1-3p expression was quantitated using real-time RT-PCR, and normalized to expression of MIR361-5p, which was recommended as a housekeeping microRNA by Thermofisher. All Taqman assays were purchased from Thermofisher.

Transposon insertion sites were mapped using two divergent primers (Mar_up and Mar_dn) on the transposon by reverse PCR. Genomic DNA was extracted using the Promega Wizard kit and digested at a concentration of 4 ng/µl in a volume of 30 µl by SspI. Restriction products were diluted to 1 ng/µl and self-ligated for 16 h at 16 °C after the addition of 100U of T4 DNA ligase (NEB). Three microliters of the ligation product were used as a template for PCR amplification using oligonucleotides Mar_up and Mar_dn. PCR products were purified by gel extraction and sequenced with the T7 rpimer. The insertion site was defined as the first nucleotide of the E. faecalis OG1RF genome downstream of the inverted repeat sequence flanking the transposon.

Heart tissue was snap frozen several hours after the death of W115 and kept at −80 °C. DNA from a frozen whole blood sample was isolated with the Promega Wizard Kit. Inspection on gel indicated that DNA from all tissues was high-molecular-weight genomic DNA. We sequenced genomic DNA across three tissues across 269 SMRT Cells (Table S1) on a PacBio RSII sequencer, which generated 16,411,063 reads larger than 500 bp with an average read length of 8334 bp. This equalled an estimate of ~45× genome-wide coverage. We also generated 100× coverage with 150 bp paired-end Illumina sequencing for the W115 genome and used BWA-mem²¹ (version 0.7.17-r1188 with default settings) and GATK^{22 (link)} (HaplotypeCaller version 3.8 with default settings) to detect SVs.

Generation of transposon libraries in the Δvca0040 and ΔyghB backgrounds was performed as previously described for HaitiWT V. cholerae^{15 (link)}. Strains were conjugated to SM10λpir E. coli bearing the donor transposon vector pSC189. Due to the moderate conjugation defect of Δvca0040, we used a 5× concentration of the conjugation reactions compared to the other libraries. Reactions were plated on 245 mm² LB + streptomycin/kanamycin agar plates to isolate V. cholerae transconjugants and incubated overnight at 30 °C. Two independent Δvca0040 and ΔyghB libraries were generated, consisting of approximately 200,000 colonies each and stocked at an A₆₀₀ of ~10 in LB + 25% glycerol. For synthetic transposon-insertion sequencing analyses, a frozen aliquot of each library was thawed and used for genomic DNA extraction with the Wizard kit (Promega). DNA libraries were prepared in an identical manner to previous transposon-insertion sequencing experiments from our group and sequenced on an in-house MiSeq platform (lllumina)^{18 (link)}. Reads were trimmed, mapped, and processed as previously described with the Con-ARTIST transposon-insertion sequencing analysis pipeline using Python version 3.0 and Matlab version 9^{18 (link)}. To identify synthetic interaction loci, we used the wild type as the ‘input’ library and the mutant as the ‘output’ library during analysis.

EDTA-anti-coagulated venous blood samples were preserved at −70 °C. Genomic DNA was isolated from whole blood using the Wizard® kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Blinded genotyping of FGFR2 rs2981582, TNRC9 rs3803662, rs12443621, and LSP1 rs3817198 was carried out using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) with Mini Option2 Real Time PCR System (BioRad, Hercules, California, USA). Assays were performed with TaqMan Universal Master Mix, TaqMan probe, and 50 ng of DNA per reaction. PCR conditions were provided by the manufacturer: 5 min initial denaturation at 94 °C followed by 45 cycles of 94 °C denaturation for 15 s and 60 °C annealing/extension for 1 min.