Qubit 2.0 fluorometer

CTAB method was used to extract total genome DNA from samples according to the DNA extraction kit (#DP328, Tiangen Company, Beijing, China). The concentration and purity of the extracted bacterial DNA were detected using Qubit^® 2.0 Fluorometer (Thermo Scientific, USA). The 16S rRNA gene V4 regions were amplified used specific primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTA AT-3') with the barcode. All PCR reactions were carried out in 30 μL reactions with 15 μL of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs). The mixture of PCR products was purified with GeneJET^TM Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacture's recommendations. The library quality was assessed on the Qubit^® 2.0 Fluorometer (Thermo Scientific). At last, the library was sequenced on the Ion S5^TM XL platform and 400 bp/600 bp single-end reads were generated.

Total bacterial DNA was isolated from all 72 samples using QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The quality and quantity of DNA samples were checked by Qubit 2.0 fluorometer (Thermo Fisher Scientific, MA, USA). The amplicon PCR was carried out using the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) to amplify the V3–V4 region of the bacterial 16S rRNA gene. Following the PCR purification, the Nextera XT indexed adapters (Illumina, CA, USA) were added to the ends of the primers. KAPA HiFi HotStart ReadyMix (2X) (Roche, Basel, Switzerland) was used for all PCR reactions. The purified libraries were then subjected to quality check on Qubit 2.0 fluorometer (Thermo Fisher Scientific, MA, USA) and Agilent 2100 Bioanalyzer system (Agilent, Singapore). The libraries were then sequenced and 250 bp paired-end reads were generated on the Illumina platform using MiSeq Reagent Kit v3 (600 cycle) (Illumina, CA, USA). By sequencing all the samples in a single MiSeq run, any potential batch effects were eliminated.

Genomic DNA (gDNA) from sampled cells was isolated using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Concentration of isolated gDNA was assessed using the Qubit 2.0 Fluorometer together with the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer. The gDNA from samples of restimulated cells was isolated using the QIAamp DNA Micro Kit (QIAGEN). The concentration of those samples was measured using the DS11 Plus (DeNovix, Wilmington, DE, USA). Total RNA was extracted from sampled cells using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s spin protocol for animal cells. Homogenisation of cells was performed using QIAshredder spin columns from QIAGEN. The iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad, Hercules, CA, USA) was used for reverse transcription. For each cDNA synthesis 15 µl total RNA (≤7.5 µg) were used as template for qPCR. The RNA concentration was determined with a Qubit 2.0 Fluorometer using the Qubit RNA HS Assay Kit (ThermoFisher Scientific) in accord with the manufacturer’s protocol.

Tissue samples (20 mg) were rapidly thawed on ice and largeRNA and smallRNA fractions were extracted from the same isolate using the RNA SPLIT Kit (Lexogen) according to the manufacturer’s instructions. Briefly, samples were homogenized in an isolation buffer and phase separated using a phenol/chloroform extraction followed by a spin column-based purification procedure. All samples were aliquoted and stored at −80°C for downstream analysis. Following extraction, sample concentrations for the largeRNA fraction were analyzed using a Qubit 2.0 fluorometer (Thermo Fisher) using the Qubit™ Broad Range RNA Assay Kit. RNA integrity was assessed using the Bioanalyzer 2100 (Agilent Technologies) and samples with low RNA integrity number (RIN) values <5 were discarded and re-extracted. SmallRNA concentrations were measured using a Qubit 2.0 fluorometer (Thermo Fisher) using the Qubit™ microRNA Assay Kit.

Reverse transcription of RNA was performed using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Subsequently, double-stranded cDNA was synthesized using RNase H and DNA Polymerase I (Takara, Dalian, China) and purified using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, Dalian, China). Double-stranded cDNA was mixed with equal volume of nucleic acid elution. DNA was randomly fragmented using Covaris S220 (Covaris, Woburn, MA, USA) to an average size of 300−350 bp. The fragmented DNA was quantified using a Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific Inc., USA). Library preparation, including end repair, adaptor linking, purification, and PCR amplification, was performed using the VAHTS Universal Pro DNA Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd., Nanjing, China). The library was determined and quantified by Agilent 2100 Bioanalyzer and Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific Inc., USA). NGS was performed using the NovaSeq platform (Illumina, San Diego, CA, US) with 2×150 paired ends.

DNA was extracted from FFPE primary tumor biopsies using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA quality was evaluated based on the absorbance ratios of A260/280 and A260/230 using a NanoDropTM 2000c Spectrophotometer (Thermo Fisher, MA, USA). DNA quantity was determined using the Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). Two independent Targeted DNA sequencing panels were employed to allow the mutational profiling of 72 cancer driver genes (see Supplementary Data 1). DNA libraries that were built with GeneRead DNAseq Colorectal Cancer Panel V2 were processed and analyzed as was previously described (31 (link)). Of note, DNA libraries that were constructed with the AmpliSeqTM for Illumina Cancer Hotspot Panel v2 Kit that allow the detection of 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes, were prepared with 100 ng of genomic DNA as was previously described (40 (link)). These DNA libraries were measured using Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). All libraries were above the minimum concentration requirement of 2 nM for further sequencing in an Illumina MiSeq platform.

Library preparation and Illumina RNA-seq were performed as previously described (Mitra et al., 2021 (link)). Briefly, RNA samples were quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, United States) and RNA integrity was checked using an Agilent TapeStation (Agilent Technologies, Palo Alto, CA, United States). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, United States). Sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, United States), and quantified with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, United States) and with quantitative PCR (KAPA Biosystems, Wilmington, MA, United States). Sequencing libraries were multiplexed and clustered onto a flowcell, loaded onto the Illumina HiSeq instrument and sequenced using a 2 × 150bp Paired End configuration. Image analysis and base calling were performed using the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.