Environmental DNA was extracted from brine and salt samples. The brine samples were filtered through 0.45 μm mixed cellulose ester (MCE) membranes. The membrane is used for DNA extractions. DNA extraction protocol from salt and filtered brine were carried out by bead beating using the PowerLyzer® PowerSoil® DNA Isolation kit (DNeasy PowerLyzer PowerSoil, QIAGEN Inc., Germantown, MD) with some minor modifications. The alternate lysis protocol that is suggested for hard to lyse cells was used. DNA was quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). Quality was additionally checked by electrophoresis on an Agarose gel (1%), and DNA was stored at -80oC. Throughout the DNA extraction, preparation, and sequencing processes, a positive control, and negative control, without DNA were used to monitor for contamination. Approximately, 5–10 ng of NanoDrop quantified DNA was amplified using standard Universal primers provided by Illumina targeting the V3–V4 region of the 16S rRNA gene. The PCR products were then subsequently used for Miseq Illumina sequencing according to the manufacturer’s instructions.
Universal primer
Manufactured by Illumina
Sourced in United States
Universal primers are short, synthetic DNA sequences designed to amplify a wide range of target sequences. They serve as essential components in various molecular biology techniques, providing a standardized approach for DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (2 mentions)
Miseq sequencing, by Illumina (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Epimark 5 hmc and 5 mc analysis kit, by New England Biolabs (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Thermo Fisher Scientific (1 mentions)
Axyprep pcr cleanup kit, by Corning (1 mentions)
16s metagenomic sequencing library preparation protocol, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Microbial dna free water, by Qiagen (1 mentions)
Quant it picogreen, by Thermo Fisher Scientific (1 mentions)
Nextera xt index primers 1 and 2, by Illumina (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Overhand adapters, by Illumina (1 mentions)
Agencourt ampure xp beads kit, by Beckman Coulter (1 mentions)
8 protocols using universal primer
1
Environmental DNA Extraction and Sequencing from Brine and Salt
2
5hmC-Enrichment and Sequencing Protocol
3
Illumina-based BCR Repertoire Sequencing
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RNA from B1a cells was reverse transcribed and amplified using a Qiagen OneStep RT-PCR kit (Qiagen Inc., Valencia, CA, USA) and iRepertoire® mouse BCR heavy chain (MBHI) primers (iRepertoire Inc., Huntsville, AL, USA), following the iRepertoire user manual for the Illumina sequencing platform. Following manufacturer’s instructions in the iRepertoire manual, Illumina adapters were added using a Qiagen Multiplex PCR kit, and a 350–500 bp bands corresponding to the Ig heavy chain amplicons were gel purified using a Qiagen Gel Extraction kit. The libraries were quantified using Qubit dsDNA quant (Life Technologies) and sequenced on the Illumina MiSeq platform (paired end, 250 bp). For samples from aged mice bearing clonal leukemias, the libraries were Sanger sequenced using the Illumina universal primers as the sequencing primer. Repertoire data was analyzed using the iRweb online analysis platform provided by iRepertoire Inc.
4
16S rRNA Gene Sequencing Protocol
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The V5-V6 hypervariable regions of the 16S rRNA gene were amplified using universal primers fused with Illumina adapters sequences U789F_v56_ngs 5′-CGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAGATACCCBDGTAGTCC-3′ and U1053R_v56_ngs 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGACGACRRCCATGC-3′ (Integrated DNA Technologies). The PCR reactions were performed in 35 μL 1x AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA, USA) and 0.4 μM of forward and reverse primers (Invitrogen, Foster City, CA, USA). Microbial DNA-free water (Qiagen) was added to PCR negative controls instead of DNA. Amplicons underwent a purification step with magnetic beads using the Axy Prep PCR Clean-Up Kit (Axygen, Union City, CA, USA) and visualized in 1.5% agarose gels. Their concentration was determined with the Qubit dsDNA HS Assay Kit (Life Technologies, Foster City, CA, USA). Equal amounts of amplicons were used for sequencing library construction using the Illumina 16S Metagenomic Sequencing Library preparation protocol. The final sequencing library was sequenced with MiSeq Reagent Kit v3 (Illumina, San Diego, CA, USA) in the Illumina MiSeq platform, using 300 bp paired-end sequencing reads with an expected output of 100,000 reads per samples.
5
Fecal Microbiome Diversity Analysis
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To detect the diversity of fecal flora in each group of mice, feces from each group were collected for differential analysis. Bacterial genomic DNA sequences were obtained by DNA extraction kit (DP712, Tiangen, Beijing, China). The target fragment of 16S rRNA V4 region was amplified by universal primers (515F/806R), and the amplified products were sequenced by Illumina platform (San Diego, California, USA) to obtain ASV/OUT feature sequences. Based on ASV/OUT, α-diversity, β-diversity and species difference analyses were performed [23 (link)].
6
Fungal community profiling of cheese
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The 5.8S-ITS2 rDNA PCR libraries were prepared for each cheese sample using universal primers with Illumina overhand adapters targeting the ITS2 region. The forward primer ITS3KYO2 (5′-GATGAAGAACGYAGYRAA-3′), and the reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used for their high coverage of fungi taxon (Toju et al., 2012) (link). Each PCR product was purified with the Agencourt AMPure XP beads kit (Beckman Coulter, Pasadena, USA). A second indexing PCR was performed with the Nextera XT index primers 1 and 2 (Illumina, San Diego, USA). The PCR products were purified as indicated above. Quantifications were made with the Quant-IT Pico-Green (ThermoFisher Scientific, Waltham, USA). To constitute a library, each PCR product, after quantification, was diluted to 10 ng/μL with Tris 10 mM Tween 20 0.05% and all PCR products were mixed together. A 1% agarose gel permitted to check, if the library was free of unwanted bands, if not, a new purification was performed with AMPure XP beads. A precise quantification, by qPCR, of each sample in the library was performed using the KAPA SYBR® FAST qPCR Kit (Kapa-Biosystems, Wilmington, USA) before normalization, pooling and sequencing on a MiSeq sequencer using v3 reagents (Illumina, USA).
7
Nested PCR with UMI Tagging and Illumina Barcoding
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Two nested PCR were performed targeting both the 271-Lama2 and TRAC target sites. The Custom PCR UMI (with SQK-LSK109), version CPU_9107_v109_revA_09Oct2020 (Nanopore Protocol) was followed from UMI tagging step to the late PCR and clean-up step. Unique Molecular Identifiers are added through a PCR of 2 cycles, called UMI tagging, to ensure that each identifier comes just from one molecule. Barcodes to demultiplex by sample are added later, after the UMI tagging, in the early and late PCR. Primers for the UMI tagging were designed using UMI tagging and gene specific primers with amplicon length of around 400 bps. Primers for the early and late PCR were the Illumina Universal primer and Illumina barcoded primer 2 and 4, for 271-Lama2 and TRAC respectively (S7 Table ). The purified amplicons were then mixed in equimolar ratio and sequenced with Illumina Miseq Nano kit v2.
8
KAPA HyperPlus Library Preparation for Illumina Sequencing
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KAPA HyperPlus kit was used to prepare the sequencing library according to the manufacturer’s protocol. ∼100 ng of DNA input was taken and fragmented enzymatically. The fragments of DNA undergo end repair where the mix converts the overhangs resulting from fragmentation into blunt ends. The 3′ to 5′ exonuclease activity of the end repair mix removes the 3′ overhangs and polymerase activity fills in the 5′ overhangs. To the blunt-ended fragments adenylation is performed by adding a single ‘A’ nucleotide to the 3′ ends. Purification of the samples is done using AMPure beads and further, the DNA is enriched by PCR with 6 cycles using NEBNext Ultra II Q5 master mix, Illumina universal primer, and sample-specific octamer primers. The amplified products are cleaned up by using AMpure beads and the final DNA library was eluted in 15 uLs of 0.1X TE buffer. The fragment analysis was performed on Agilent 2100 Bioanalyzer, by loading 1 uL of the library into Agilent DNA 7500 chip. The sequence used for the whole genome analysis was Illumina Hisq 4000.
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