Geneamp 9700 thermocycler

The selection criteria for large scale genotyping of genetic variants identified from direct sequencing were as follows: for less common variants (minor allele frequency in these subjects between 0 to 10%), > 2% difference in minor allele frequency between normal controls and the patients with schizophrenia; for common variants (minor allele frequency was between 10% to 50%), > 4% difference in minor allele frequency. All SNP genotyping was performed using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) [35 (link)]. Primers and probes flanking the SNPs were designed using SpectroDESIGNER software (Sequenom, San Diego, CA, USA). A DNA fragment (100–300 bp) encompassing the SNP site was amplified by PCR (GeneAmp 9700 thermocycler, Applied Biosystems, CA, USA) according to the manufacturer’s instructions.
After removing the un-incorporated deoxynucleotide triphosphate (dNTP) and inactivating the shrimp alkaline phosphatase from the PCR product, primer extension was performed by adding the probe, Thermo Sequenase (Amersham Pharmacia, Piscataway, NJ, USA) and an appropriate dideoxynucleotide triphosphate (ddNTP/dNTP) mixture, followed by 55 cycles of denaturing at 94°C for 5 sec, annealing at 52°C for 5 sec, and extension at 72°C for 5 sec. The other extension products were differentiated by mass through MALDI-TOF [36 (link)].

The copy numbers of CHD7 mRNA were evaluated using droplet digital PCR (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, cDNA was synthesized from 5 ng total RNA extracted from cells cultured with hPSCs or Es8 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher). The PCR mixture was loaded into a Bio-Rad QX-100 emulsification device, and droplets were formed following the manufacturer’s instructions. cDNA was amplified separately in an Applied Biosystems GeneAmp 9700 Thermocycler. Each 20-µL reaction contained 10 µL droplet digital PCR (ddPCR) Probe Supermix, 1000 nM primers, 250 nM probe, and template cDNA, and PCR was carried out using the following cycling conditions: 10 min at 95 °C, 40 cycles of 30 s denaturation at 94 °C and 60 s of annealing/extension at 53 °C, and a final incubation for 10 min at 98 °C. After cycling, raw fluorescence data for each well were exported from the manufacturer’s software (Bio-Rad QuantaSoft v. 1.2) for analysis.

DNA was extracted from both tick and human samples using the Qiagen DNeasy Blood and Tissue kit (Qiagen Inc., Valencia, CA). The DNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher scientific) and stored at -70 to -80°C. Coxiella burnetii was detected using a single step conventional PCR assay using the primers Trans 1 and Trans 2 [4 ] designed to amplify a 687-bp fragment of the repetitive, transposon-like IS1111 region. The PCR amplification conditions for the Trans primers included an initial denaturation step at 95°C for 2 min, followed by five cycles at 94°C for 30 s, 66 to 61°C (the temperature was decreased by 1°C between consecutive steps) for 1 min, and 72°C for 1 min. These cycles were followed by 35 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min and then a final extension step of 10 min at 72°C. Coxiella burnetii DNA was used as a positive control and water was used as a negative control. The PCR assays were performed in a Gene Amp 9700 thermocycler (Applied Biosystems) using a Taq PCR master mix kit (Qiagen Inc., Valencia, CA), 1ng of template DNA, and 1μl of 50 μM of the Trans primer in a 25μl reaction mix. PCR products were separated on a 2% agarose gel visualized with ethidium bromide on a UV transilluminator. Products were sized using an O'rangeRuler 100bp DNA ladder (Thermo Fisher Scientific).

Total DNA was extracted from whole individuals of Bythotrephes and potential prey species using the QIAGEN DNeasy Blood & Tissue Kit (Valencia, CA, USA) following manufacturer protocols. DNA was eluted in 100 μL of AE buffer and stored at −20°C. Following genomic DNA (gDNA) extraction, purified DNA was quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and a Qubit dsDNA High Sensitivity Assay Kit (Life Technologies, Eugene, OR, USA). Amenability of the extracts for PCR amplification was determined by amplifying 18S rRNA using universal primers Univ-18SF-577F (5′ CAG CAG CCG CGG TAA TTC C 3′) and Univ 18S-1180R (5′ CCC GTG TTG AGT CAA AAG C 3′) as previously described (Frischer et al., 2017 ). Amplification was accomplished using a GeneAmp 9 700 Thermocycler (Applied Biosystems, Foster City, CA) and included a 3 min initial denaturation at 95°C followed by 30 amplification cycles [94°C (30 s), annealing temperature of 60°C for 30 s, 72°C (1 min)] followed by a 10 min final extension step at 72°C.

To identify species, PCR amplification of the 16S rRNA gene was performed with the following primers: 27F, 5¢-AGA GTT TGA TCM TGG CTC AG-3¢ and 1088R, 5¢-GCT CGT TGC GGG ACT TAA CC-3¢ [15 ]. PCR was performed in a GeneAmp 9700 thermocycler (Applied Biosystems, USA) with the following thermal cycling conditions: pre-denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 sec, 57°C for 30 sec, and 72°C for 45 sec, followed by 10 min at 72°C. DNA fragments were purified using a QIAquick Gel Extraction Kit (Qiagen, USA) in accordance with the manufacturer’s instruction. Sequence reactions were performed with an ABI 3730XL DNA analyzer (Applied Biosystems) by Bionix (Korea). Sequences were analyzed using the BLAST algorithm at the National Center for Biotechnology Information web server (https://www.ncbi.nlm.nih.gov).

Total RNA was isolated from snap-frozen human GBC tissue and NNT using TRIzol Reagent (Life Technologies; Woolston, UK) followed by phenol–chloroform extraction. siRNA-transfected cells were washed three times with PBS, scraped off with PBS and lysed with TRIzol Reagent. Total RNA (1 µg) was reversely transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA) according to the manufacturer´s instructions (GeneAmp 9700 Thermocycler, Applied Biosystems; Foster City, USA). For gene expression analyses, the Power SYBR Green PCR Master Mix Kit (Applied Biosystems, Foster City, USA) was used in a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, USA). GAPDH was found to be the most stable endogenous control using NormFinder, and relative gene expression levels were calculated using the 2^∆∆CT method.