Mastercycler gradient

Routine blood data was obtained from pregnant women in the first trimester (0–12 weeks), fasting venous blood for about 3 mL. The full blood count was completed by Sysmex XN-1500 automatic blood analyzer in the Laboratory Department of the First People's Hospital of Chongqing Liang Jiang New Area, the evaluation included: WBC, RBC, Hb, HCT, MCV, MCH, MCHC, RDW-CV, RDW-SD, and PLT.
The characteristics of thalassemia were determined by DNA detection. 2 mL of fasting venous blood was drawn from pregnant women, from which three common deletion α-thalassemia genes (-SEA, -α3.7, -α4.2) were detected with the gap-PCR amplification method, and eight common sites and nine uncommon sites of β-globin gene mutation were detected by PCR-membrane reverse dot hybridization technique. The detection instrument is Eppendorf Mastercycler Gradient. The parallel joint analysis was used: any positive result of the two indicators is determined positive.
Pregnant women who carried the thalassemia gene and serum ferritin (SF) ≥ 20 μg/L were divided into the study group, and those without the thalassemia gene and serum ferritin (SF) < 20 μg/L in the control group [22 (link)].

At confluence, PMVEC or PAEC lysate, and whole kidney and stomach tissue was prepared using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and lysates stored at -80°C. Following extraction, total RNA was treated with DNase I to remove genomic DNA contamination. The concentration and integrity of the RNA samples was assessed by measuring spectral absorption at 260 and 280nm using NanoDrop Lite spectrophotometer (Thermo Scientific). An A_260/A280 estimate of 1.8–2.2 was accepted. cDNA was synthesized from 1 μg of DNA-free RNA template in a 20 μL reaction volume using iScript cDNA Synthesis Kit (catalogue no 1708891, Bio-Rad). The complete reaction mixture was incubated in a Mastercycler gradient (Eppendorf) at 25°C for 5 min followed by 20 min at 46°C for reverse transcription. The reverse transcriptase was inactivated by heating to 95°C for 1 min. Samples of cDNA were stored at -20°C.

The Total tumour RNA were isolated using Trizol^® reagent (Invitrogen, Saint Aubin, France) according to the manufacturer’s protocol, and quantified using a Nanodrop spectrophotometer (Nanodrop^®2000, Thermo scientific, Waltham, MA, USA). Reverse transcription was performed on 1 μg of total RNA with the high-capacity cDNA kit (Applied Biosystems, Saint Aubin, France) using random hexamer (pdN6) primers in a thermocycler (Mastercycler^® gradient, Eppendorf, Montesson, France).
Quantification by qPCR was performed using SYBR^®Green reagent according to the manufacturer’s instructions on a StepOne system (Applied Biosystems, Saint Aubin, France). All primers used are summarized in Table S3. Each sample was assayed in triplicate. Relative quantification was obtained by the comparative CT method, based on the formula 2^−ΔΔCT [19 (link)]. GAPDH was used for normalizing data. Tbx21, Gata3, Foxp3, Cd8α, DX5, CD1d, perforin 1, granzyme A and B levels were expressed as the fold difference between diet groups.

1-mm-thick frontal sections of fresh brain tissue were sectioned with razor blades in cooled PBS (4°C). Each section was divided into four parts of equal size by a horizontal and a vertical cut. These parts were individually shock-frozen in reaction tubes on dry ice and stored at −70°C. RNA was isolated using the GenElute Total RNA purification kit (Sigma-Aldrich, St. Louis, MO, United States). 1 μg RNA was used for cDNA synthesis with the First strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, United States). Details of the PCR buffers, primer sequences and the PCR program are provided in Supplementary Table 4. In short, PCR was performed with 1 μl of cDNA in a Mastercycler gradient (Eppendorf, Hamburg, Germany). The number of cycles was reduced from a standard of 25 to 20 for β Actin samples to avoid saturation effects. DNA amplicons were separated by agarose gel electrophoresis according to their size. Gels were documented with a digital camera under UV light (LTF, Wasserburg am Inn, Germany). Band intensity was measured densitometrically with ImageJ: after background subtraction, a box of equal size was drawn around each band and the “mean” value was determined using the “Measure” tool. Each value was normalized to the housekeeping gene β Actin. Data were analyzed with Excel software and visualized with Adobe Illustrator.

Isolation of total RNA from mycelium of P. sapidus at culture day six and cDNA synthesis were performed as described previously [13 (link)] using the primer 5′-AAGCAGTGGTATCAACGCAGAGT ACGCTTTTTTTTTTTTTTTTTTT-3′ for reverse transcription. Specific primers for gene amplification were deduced from the ORF-start (P1: 5′-ATGACTACACCTGCACCACCCCTCGACCTC-3′) and -stop (P2: 5′-TCAAGCAGAGATTGGAGCTTGGGTSWGAGGA-3′) region of the homologous peroxidase of Pleurotus ostreatus PC15 (GenBank accession no. KDQ22873.1). PCRs were performed with Phusion High-Fidelity DNA Polymerase and the Master Cycler gradient (Eppendorf, Hamburg, Germany) as described elsewhere [45 (link)]. The cycler program was as follows: denaturation for 2 min at 98 °C, 35 cycles at 98 °C for 1 min, 62 °C for 30 s and 72 °C for 90 s, and a final elongation at 72 °C for 10 min. Analysis of PCR products, ligation, transformation in Escherichia coli, colony PCR, and sequencing were performed as described by Behrens et al. [13 (link)]. Translation of DNA sequences was performed using SnapGene^® (GSL Biotech LLC, Chicago, IL, USA). Sequence homology was examined using BLAST [46 (link)]. Alignments were produced by ClustalOmega [47 (link)].

Total RNA was extracted using the RNeasy® Mini or Midi kit (Qiagen, Hilden, Germany) and digested with Rnase-free DNaseI (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The quality and quantity of RNA was assessed photometrically using the BioSpectrometer® (Eppendorf, Hamburg, Germany). To obtain cDNA, 1 μg of RNA was used for reverse transcription with a cDNA synthesis kit and random hexamer primers (Fermentas GmbH, St. Leon-Rot, Germany). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using 1 μl cDNA using Taq DNA polymerase (JumpStart™ Taq DNA polymerase (Sigma-Aldrich) in a Mastercycler Gradient (Eppendorf, Hamburg, Germany) with primer pairs listed in Table S4 (Sigma-Aldrich). The RT-PCR conditions were as follows: 94°C for 2 minutes 30 seconds (initial denaturation), 25-35 cycles of 94°C for 30 seconds (denaturation), 60°C for 45 seconds (annealing), and 72°C for 60 seconds followed by 72°C for 5 minutes (final elongation). Samples were documented as described above.