Maxwell rsc instrument

For generation of E. coli Illumina data sets, the Genomic-tip 20/G kit DNA extraction was used, as described elsewhere (24 (link), 25 (link)). The DNA extraction of the E. coli ONT data sets was performed using the Maxwell RSC Instrument (Promega, WI, USA) according to the manufacturer’s instructions. For the Illumina and ONT sequencing of the L. monocytogenes isolates, genomic DNA was also extracted from pure bacterial cultures using the Maxwell RSC Instrument (Promega), except for the Illumina sequencing of the L. monocytogenes isolates collected from humans, for which the MagCore Genomic DNA Bacterial Kit (RBC Bioscience, New Taipei City, Taiwan) was used instead. DNA concentration and purity were assessed using the dsDNA high-sensitivity and broad-range assay kits for the Qubit 4 fluorometer (Thermo Fisher Scientific, Schwerte, Germany) and the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), respectively. All kits were used according to the manufacturer’s instructions.

For RT-qPCR gene expression analysis, cDNA samples were prepared from leaf DNA-free RNA obtained using the Maxwell RSC Plant RNA Kit with the Maxwell RSC Instruments (Promega) according to manufacturer’s instructions. Real-time PCR assays were performed in triplicate with SYBR Green I Master (Roche Diagnostics) using a Light Cycler 480 detection system (Roche Diagnostics) as described in Ramirez-Estrada et al. (2017) (link). Specific primer pairs for target genes SlPSAT1, SlASAT1 and SlSAG12 (Solyc02g076910), and the actin gene (Solyc03g078400) used as reference gene, are shown in Supplementary Table 1.

Total RNA was extracted from the FFPE (Formalin-Fixed-Paraffinn-Embedded) samples from 17 matched tumors from patients before and after the development of resistance to BRAF inhibitors as single agent or in combination with MAPK inhibitors (see Table 2). MiRNAs were extracted from FFPE melanoma tissues before the beginning of therapy and at disease progression through Maxwell^® RSC RNA FFPE Kit (Promega, Madison, WI, USA) and processed with Maxwell^® RSC Instruments (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Real-time PCR was assayed as described above. The use of human samples was approved by the Ethics Committee of Istituto Nazionale Tumori—IRCCS—Fondazione "G. Pascale", Naples, Italy (date of registration 18/01/2018; project identification code n° 33/17 oss, 2018. Written informed consent was obtained from all participants.

The human BTC tissues were collected under approval of the Ethical Committee for Clinical Research at Azienda Ospedaliera Universitaria, Modena, Italy (ID 465/2018/SPER/AOUMO). The study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as per ethical approval given by the institutional review board. Written informed consent was obtained from all participants. Diagnosis of BTC was established on radiological findings and was pathologically proven in all patients. Inclusion Criteria, Subject Demographics, Sex and Biological Variables and Clinicopathological characteristics were shown in Table S1.
Disease recurrence was defined as the presence of imaging‐proven disease.
Total RNA was extracted from the FFPE (Formalin-Fixed-Paraffinn-Embedded) samples from 62 tumor and the matched nontumor component after microscopic dissection (Table S1). MiRNAs were extracted from FFPE tissues before the beginning of therapy and at disease progression through Maxwell® RSC RNA FFPE Kit (Promega, Madison, WI, USA) and processed with Maxwell® RSC Instruments (Promega, Madison, WI, USA) according to the manufacturer’s instructions [21 (link), 22 (link)].

Genomic DNA used in SNP genotyping (CottonSNP63K array and KASP) and re-sequencing was extracted from young true leaves using a DNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions or the cetyl trimethylammonium bromide (CTAB) method with home-made solutions. NanoDrop 1000 (Thermo Scientific) was used in the quantification of DNA concentration. DNA samples used in next-generation sequencing were also quantified using Qubit (Invitrogen) and checked for integrity by gel electrophoresis.
Total RNAs were extracted from 0, 1, 2, 3, 4, 5, and 6 dpa ovules of fuzzless-tufted and normal fuzzy seed NILs using the Maxwell RSC Instrument (Promega) with a Maxwell RSC Plant RNA Kit. RNA quality was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies) and Qubit (Invitrogen). RNA samples from 0, 2, 4, to 6 dpa with an RNA integrity number (RIN) above 7.0 were used in RNA-sequencing, while samples from all seven time points were used in qRT-PCR analysis. Each sample was analyzed with three biological replicates.

Genomic DNA was isolated from EDTA blood using the Maxwell^® RSC Whole Blood DNA Kit with the Maxwell^® RSC instrument (Promega, Dübendorf, Switzerland).