Maxwell 16 research system

Two million HuH7 cells were incubated in 100 μl of cytoplasmic buffer (50 mM Tris HCl pH7.4, 1 mM EDTA, and 1% NP40) for 5 min at 4°C. Then, cells were centrifuged for 5 min at 3000 g and the supernatant was used to isolate cytoplasmic RNA. The pellet was washed with cytoplasmic buffer and centrifuged as before. The supernatant was discarded and the pellet was used to isolate the nuclear RNA. RNA from nuclear and cytoplasmic fractions was isolated with MaxWell 16 research system (Promega).

Human tissue was homogenized using the ULTRA-TURRAX dispersing machine (t25 basic IKA-WERKE) (51 (link)). Total RNA from the tissue was extracted in 1 ml TRIZOL (Sigma-Aldrich) and recommendations of the supplier were followed (52 (link)). DNase (Fermentas) treatment was performed to eliminate DNA from the samples before RT-PCR reactions. RNA was extracted from cells with the MaxWell 16 research system from Promega following the manufacturer’s recommendations. RNA concentration was measured using NanoDrop 1000 Spectrophotometer. The quality of the RNA was analyzed by Bioanalyzer (Agilent Technologies). Reverse Transcription (RT) was performed as described (53 (link)). The reaction was performed in the C1000 Touch Thermal Cycler from Bio-Rad. The samples were incubated at 37°C for 60 min, then at 95°C for 60 s and then immediately cooled to 4°C. qPCR was performed in the CFX96 Real-Time system from Bio-Rad as described (54 (link)). The results were analyzed with Bio-Rad CFX-manager software. GAPDH levels were evaluated in all the cases as a reference. Only the samples with similar GAPDH amplification were analyzed further. The primers used are listed in Table S2 in Supplementary Material and were designed with the Primer3 program¹.

Tumor areas were marked on an H&E stained slide and corresponding tissue areas were microdissected from three subsequent unstained slides. Tumor cell content was documented, for later correction of allele frequencies. Extraction of genomic DNA was performed by proteinase K digestion and fully automated purification using either the QIA Symphony SP (Qiagen, Hilden, Germany) or the Maxwell 16 Research System (Promega, Madison, USA). DNA content was measured fluorimetrically using the QuBit HS DNA Assay (Life Technologies) and DNA sequencing grade quality was confirmed using a real-time qPCR-based method (RNAseP Detection system, Life Technologies).

The sex of all Kori bustards was determined from collected feathers. The superior umbilicus and the tip (5–10 mm long) of the calamus of each feather were placed in a 2 mL Eppendorf tube, which contained 470 μL lysis buffer ATL (Qiagen, Hilden, Germany) and 30 μL Proteinase K (Qiagen). The feathers were digested overnight in an incubator at 56°C and pulse-vortexed twice during that period. Genomic DNA was extracted using the Maxwell 16 Research System (Promega, Madison, WI, USA) and the Maxwell 16 tissue DNA Purification Kit following the manufacturer’s protocol.
Sex was determined using the universal primers P2 and P8, which target the sex-linked chromobox-helicase-DNA-binding (CHD) genes CHD-W and CHD-Z [30 (link)]. The P8 primer was fluorescently (6FAM) labelled. A polymerase chain reaction (PCR) was performed with Qiagen’s Multiplex PCR Kit following the manufacturer’s protocol but used an 8.4 μL reaction volume. PCR products (1 μL) were mixed with a 0.14 μL GeneScan 500 LIZ (Applied Biosystems, Foster City, CA, USA) size standard and 6.16 μL Hi-Di formamide following capillary electrophoresis on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Allele sizes were assigned using GeneMapper v5.0 software (Applied Biosystems). A single band (379 base-pairs) was amplified in males, and two bands (379 and 396 base-pairs) were amplified in females.

The genomic DNA of selected LAB strains, potential producers of therapeutic biomolecules, was extracted using a Maxwell^® DNA Cell kit in a Maxwell^® 16 Research System instrument (Promega, Madison, WI, USA). WGSwas conducted on an Illumina MiSeq 2500 platform (Illumina, San Diego, CA, USA) at IGA Technology Services (IGA Technology Services Srl, Udine, Italy) using a paired-end approach [53 (link)]. This Whole Genome Shotgun project was submitted to the NCBI nt database (BioProject PRJNA388578, version 1.0) under the accession numbers listed in the Table 4 for each strain with the following feature: biosynthesis of S-layer proteins, plantaricin and EPSs [22 ].