Maxwell 16 system

The enrichment of the hydrocarbon-oxidizing bacteria was then used to isolate and characterize the microorganisms. Serial dilutions (10⁵–10⁷) of the suspension were prepared in sterile water and then plated on LB and R2A agar and incubated at 30 °C for 5 days. Several colonies were visualized but with a few different phenotypes, consistent with a recent contamination. After a few passages of plate streaking, 20 pure colonies were selected. Genomic DNA was extracted with a Maxwell 16 system (Promega^®), and the 16S rRNA fragments were amplified via polymerase chain reactions (PCR) [48 (link)].
A BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc. 850 Lincoln Centre Drive Foster City, CA 94404 USA) and the same primers used in the previous PCR were used to sequence the PCR-amplified 16S rRNA with an automated DNA sequencer (SEQ Studio Genetic analyzer).
A SeqMan application from Lasergene v. 16.0.0 (DNASTAR, Inc.3801 Regent St.Madison, WI 53705 USA)was employed to edit and assemble the nucleotide sequences obtained. Then, a homology comparison was performed (basic local alignment search tool, BLAST, analysis), exploiting the information available at the National Center for Biotechnology Information server (NCBI, www.ncbi.nlm.nih.gov/blast/Blast.cgi, accessed on 18 February 2022) [49 (link)].

Tissue samples were pooled since they were from the same species. A total of 5 mg fragments of the pooled tissues was homogenized in a tube filled with 600 µL of 1-Thioglycerol/Homogenization Solution and one 5 mm tungsten bead using the TissueLyser II system (Qiagen, Hilden, Germany) for 2 min at 25 Hz. RNA extraction was performed with a Maxwell^® 16 LEV simplyRNA Tissue Kit (Promega, Madison, WI, USA) in the Maxwell^® 16 System (Promega) according to the manufacturer’s protocol and was followed by double strand cDNA (complementary DNA) synthesis applying the SuperScript^TM VILO^TM Master Mix (Thermo Fischer Scientific, Waltham, MA, USA) for first strand synthesis and the NEBNext^® mRNA Second Strand Synthesis Module (New England BioLabs, Ipswich, MA, USA) for second strand.

The homogenization of tissue samples and total RNA extraction from tissue were performed in an automated Maxwell 16 system (Promega, USA) using the Maxwell 16 total RNA purification kit (Promega, USA) according to the manufacturer’s instruction. Following the extraction, RNA of the sample was reverse transcribed into complementary DNA (cDNA) using the ImProm-II™ reverse transcription system (Promega, USA).

After careful manual microdissection, DNA was analyzed from FFPE tumor tissue using the Maxwell© 16 system (Promega, Madison, WI, USA) according to manufacturer’s instructions. DICER1 sequence analysis was performed using the QIAseq Targeted Human Comprehensive Cancer Panel according to manufacturer’s instructions (the list of the 160 genes is shown in the supplementary file). Bioinformatic evaluation of the sequencing data, including variant calling and annotation, was done with the CLC Genomics Workbench (QIAGEN, Redwood City, CA, USA). Low-quality variants with a score under 200 were filtered out, as well as variants in non-protein-coding regions, synonymous variants, and those present in GnomAD with an allele frequency of over 1%. The remaining variants were assessed for pathogenicity according to ACMG/AMP criteria. The DICER1 variants were classified as described previously [6 (link)].

Total RNA was extracted from approximately 10 mg of five placentomes using the commercial Maxwell^® 16 LEV simplyRNA Purification Kit, developed for automated Maxwell^® 16 System (Promega, Wisconsin, USA), following the manufacturer’s recommendations. It included a DNAse treatment step to avoid genomic DNA contamination of the RNA samples. For all samples, RNA concentrations were determined by spectrophotometry (Nanophotometer, Implen), and the integrity of the RNA was checked by the 260/280 absorbance ratio (close to 2.0) and the visualization of the 18S and 28S ribosomal subunits after electrophoresis on a 1% agarose gel.
Reverse transcription was carried out by the master mix SuperScript^® VILO™ cDNA Synthesis Kit (Invitrogen, Paisley, UK) in a 20 μL reaction using up to 2.5 μg of total RNA. Obtained cDNA reactions were diluted 1:10 and used in quantitative PCR (qPCR) assays.

DNA from the following 20 colon cancer cell lines was used to investigate promoter methylation status; Co115, Colo205, Colo320, EB, FRI, HCT15, HCT116, HT29, IS1, IS3, KM12, LS1034, LS174T, NCI-H508, RKO, SW480, SW620, SW948, TC71, and V9P. HCT15, HCT116, NCI-H508, RKO, SW620, and SW948 have been purchased from the American Type Culture Collection (ATCC), and Colo205 and KM12 from the Charles River Laboratories. The rest of the cell lines have kindly been provided by Professor Richard Hamelin (INSERM, Paris, France). DNA was isolated using either a standard phenol/chloroform protocol, or a magnetic beads approach (Maxwell^® 16 System, Promega). For each cell line, the same DNA stock was used for all analyses. All cell lines have been tested by short tandem repeat (STR) profiling using the AmpFLSTR Identifiler PCR Amplification Kit (Life Technologies). Commercial lines have been authenticated based on comparisons with available STR profiles from the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell cultures13 (link). All cell lines tested negative for mycoplasma infection (MycoAlert Mycoplasma Detection Assay and Lucetta Luminometer, Lonza).

Total RNA was automatically isolated by Maxwell® 16 system (Promega; Mannheim, Germany), and cDNA was obtained by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA, USA). Real-time polymerase chain reaction (qPCR) was carried out on a 7300 Real-Time PCR System, using the TaqMan® chemistry with commercial primers and probes (BAX, Hs01016552_g1; BCL2, H Hs00608023_m1) (Applied Biosystems; Foster City, CA, USA). Gene expression was normalized to the reference gene β-actin (4326315e). Data were analyzed with 7300 System SDS Software (Applied Biosystems; Foster City, CA, USA), and are presented as mean fold change of expression compared to control cells, normalized to one.