Qubit2 fluorometer

Frozen sections (10 μm in thickness) of tumor and nontumorous tissues were fixed in ice‐cold 4% formalin for 10 min, washed by water for 5 min, and subsequently stained by hematoxylin. Tumor and non‐tumorous cells were collected from the sections by laser microdissection (LMD) using LMD 7000 (Leica Microsystems, Inc., Bensheim, Germany). Genomic DNA was extracted from the collected cells using a QIAamp DNA formalin‐fixed, paraffin‐embedded tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Concentration of DNA was assessed by e‐SPECT (Malcom, Tokyo, Japan) and Qubit2 fluorometer (Invitrogen, CA). All genomic DNA was stored at −20°C until use.

Total RNA was isolated from CD138+ plasma cells, using TRI Reagent^® (Molecular Research Center Inc., Cincinnati, OH, USA). The concentration of each total RNA was determined using Qubit™ 2 Fluorometer (Invitrogen™, Thermo Fisher Scientific Inc., Carlsbad, CA, USA), and its integrity was assessed by agarose gel electrophoresis. Next, 200 ng of each total extract were subjected to in vitro polyadenylation, using E. coli poly(A) polymerase (New England Biolabs Ltd., Ontario, ON, Canada) and 80 μM ATP. Each sample was incubated at 37 °C for 60 min, followed by an inactivation step at 65 °C for 10 min. Reverse transcription was then performed, using MMLV reverse transcriptase (Invitrogen™, Thermo Fisher Scientific Inc.) and an oligo-dT–adapter primer, following the manufacturer’s instructions. The oligo-dT–adapter primer sequence was 5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′. Both polyadenylation and reverse transcription were conducted in a GeneAmp^® PCR System 9700 (Applied Biosystems™, Thermo Fisher Scientific Inc., Foster City, CA, USA).

Sample data presented in Fig. 1 were extracted using an EZNA tissue DNA kit (Omega Bio-Tek Inc., USA) according to the manufacturer’s protocol. Prior to extraction, gill wash samples were defrosted on ice, and 10 mL wash buffer was filtered through a 0.2-μm-pore-size syringe filter (Whatman International Ltd., United Kingdom). The filter was extricated and used for DNA extraction. Gill filter swabs were extracted directly from the filter. Whole tissue was homogenized using a bead beater and lysis kit buffer. Following lysis, samples were bead beaten for 1 min. To increase the efficiency of DNA extraction from Gram-positive bacteria, with their prominent feature of thick peptidoglycan cell walls, samples were heated to 95°C for 10 min as per Knudsen et al. (7 (link)). Standard kit instructions were followed for DNA eluted from columns using 50 to 200 μL elution buffer. DNA purity and concentration were evaluated using a Nanodrop ND-1000 spectrophotometer (ThermoFisher Scientific, United Kingdom) and Qubit 2 fluorometer (Invitrogen, USA). Sample data presented in Fig. 2 to 4 were extracted as described above, but we used a Tris-EDTA lysis buffer following the methods described by Longmire et al. (38 ).

The purity, concentration and integrity of RNA samples were detected using a Nanodrop spectrophotometer (BioRad, Philadelphia, PA, USA), a Qubit 2 fluorometer (Invitrogen, Carlsbad, CA, USA) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively, to ensure that qualified samples were sequenced. For Illumina sequencing (Illumina, San Diego, CA, USA)), a sRNA library was constructed using the small RNA Sample Pre Kit as well as the sRNA passing quality test. Briefly, the small RNA (18–30 nt) was first connected to a 3′ adaptor; primers for the reverse reaction were added; and a 5′ adaptor was ligated to the small RNA. Finally, a reverse transcription reaction was followed by several cycles of PCR to obtain sufficient product for sequencing, and the target fragments were screened by PAGE gel electrophoresis. The small RNA library was constructed using all sequences from the fragments..

Ion torrent deep amplicon sequencing of tumor samples: deoxyribonucleic acid was extracted from tumor tissues and quantitated using the Qubit2 fluorometer (Invitrogen, Grand Island, NY). Ten nanograms of DNA was used for multiplex PCR of a panel covering 739 mutations in 46 cancer-related genes (Ion AmpliSeq Cancer Panel, Life Technologies, Grand Island, NY). Subsequent processing of samples was performed according to the manufacturer's protocol. Library constructions of the amplicons and subsequent enrichment of the sequencing beads was performed using the OneTouch (Grand Island, NY) system. Sequencing was done on the 314 chip with 10 megabases capacity using the Ion Torrent Personal Genome Machine (Life Technologies) as per the manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome and base calling, was done using built-in software.

Between 50–100 mg of each powdered sample was weighed directly into a clean 1.5 ml Eppendorf tube. Initial lysis of samples was conducted in 360 µl of ATL buffer + 20 µl of Proteinase K (20 mg/ml) per reaction by incubation at 56 °C and 900 rpm overnight. DNA extractions were performed using the Qiagen QIAamp Investigator Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions for DNA extraction from bone. In the final elution step, DNA was eluted from Qiagen membranes in 25 µl of ATE buffer and stored at −20 °C until further processing. Negative controls (i.e., no template DNA PCR and no sample DNA extraction) were performed to account for contaminants in bacterial community analysis. Quantification of DNA was conducted using a Qubit 2-Fluorometer (Invitrogen, Waltham, MA).