For cDNA synthesis, extracted mRNA using the RNAeasy mini kit (Qiagen) of flow cytometric sorted Vγ9+ and Vγ9− cells, 5 µL mRNA of both subsets was pooled equally for analysis of total γδ T cells. CDR3 TRG and TRD sequencing amplicons were generated as described previously (19 (link)), while using 25–30 PCR cycles. According to Illumina guidelines 96 samples were labeled with Nextera XT indices and subjected to Illumina MiSeq analysis using 500 cycle paired-end sequencing. 20% PhIX was added as an internal control and to increase library complexity. Illumina output fastq files were processed using ea-utils.
Nextera xt indices
Manufactured by Illumina
Sourced in United States
The Nextera XT indices are a set of oligonucleotide sequences used in the Nextera XT DNA library preparation workflow for next-generation sequencing. These indices enable the multiplexing of multiple DNA samples in a single sequencing run, allowing for efficient and cost-effective high-throughput sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (5 mentions)
Miseq platform, by Illumina (4 mentions)
Bioanalyzer dna 1000 chip, by Agilent Technologies (4 mentions)
Miseq sequencer, by Illumina (2 mentions)
Qiaquick gel purification kit, by Qiagen (2 mentions)
Qubit dsdna system, by Thermo Fisher Scientific (2 mentions)
16s metagenomic sequencing library preparation guide, by Illumina (2 mentions)
Agencourt ampure xp, by Beckman Coulter (2 mentions)
Fluorometric quantification method, by Agilent Technologies (2 mentions)
Miseq system, by Illumina (2 mentions)
Sp reagent kit, by Illumina (2 mentions)
Mid output reagent kit, by Illumina (2 mentions)
Nextera flex, by Illumina (2 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Dneasy powersoil htp 96 kit, by Qiagen (1 mentions)
Picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Neb q5, by New England Biolabs (1 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
Nextseq 500 550 mid output kit v2, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
24 protocols using nextera xt indices
1
cDNA Synthesis and Sequencing of γδ T Cells
2
Longitudinal 16S rRNA Profiling of Mouse Gut Microbiome
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The 16S rRNA profiling was performed on mouse fecal pellet samples collected from Days −1, 8, 29, 57 and 79. Using sterile forceps, mouse fecal pellets were placed into the wells of a Bead Plate from the DNeasy PowerSoil HTP 96 Kit (Qiagen, Hilden, Germany), and DNA were extracted by using the manufacturer’s protocol. The variable V4 region (515F/806R primers) was amplified by PCR under the following conditions: 10 min at 95 °C followed by 35 cycles of 95 °C 15 s, 55 °C 30 s, and 72 °C for 2 min. PCR products were purified using AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) and Nextera XT indices (Illumina, San Diego, CA, USA) were added during a second PCR. Samples were pooled, quantified using the PicoGreen DS DNA Assay (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced for 2 × 250 cycles on the MiSeq (Illumina, San Diego, CA, USA) per the manufacturer’s instructions. The resulting paired-end reads were merged using FLASH [33 (link)], primers were removed, and reads with an overall quality score less than 20 were discarded by the RDP Initial Process tool [23 (link)]. The reads were clustered at 99% similarity by CD-HIT [34 (link)] and those with abundance >0.1% were assigned taxonomy against the RDP 16S rRNA taxonomy training set no. 18 [35 (link)].
3
Microbial Profiling by 16S rRNA
4
Gut Microbiome Profiling with 16S rRNA
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Fresh fecal samples were collected into a sterile container from each volunteer enrolled in the study, immediately frozen at −20 °C after defecation, and stored at −70 °C for 24 h until further manipulation. Total DNA was extracted from stool samples within 1 month of storage using the MOBio PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. To amplify and sequence the V3-V4 hypervariable region of the 16S rRNA gene, specific fusion primers were used (Illumina, San Diego, CA, USA). Libraries were pooled for sequencing using the full complement of Nextera XT indices and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions [9 (link)].
5
Whole Genome Sequencing of Microbial DNA
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Genomic DNA was extracted using the MN NucleoSpin®Microbial DNA (Machery-Nagel GmbH and Co.KG, Duren, Germany) kit following the manufacturer’s instructions. The quantification of extracted genomic DNA was determined using a DeNovix DS-11 + Spectrophotometer. WGS was conducted at a high-throughput sequencing facility at SA Pathology in South Australia. Sequencing libraries were prepared using the Nextera XT DNA library preparation kit (Illumina Inc., San Diego, Ca, USA), with modifications of the kit’s protocol. Briefly, genomic DNA was fragmented, followed by the amplification of Nextera XT indices (Illumina Inc., San Diego, CA, USA) to the DNA fragments using a low-cycle PCR reaction. The amplicon library was then purified, and normalised manually. Whole-genome sequencing (WGS) was performed on the Illumina NextSeq 550 platform with NextSeq 500/550 Mid-Output kit v2.5 (300 cycles) (Illumina Inc., San Diego, Ca, USA).
6
Bacterial 16S rRNA Gene Sequencing Protocol
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Bacterial 16S rRNA gene amplification and library construction was performed according to the 16S Metagenomic Sequencing Library Preparation guide from Illumina (Forest City, CA, USA) with minor modifications. All beads, tubes and non-enzymatic reagents were treated with UV light for 30 min prior to use (Tamariz et al. 2006 (link)). Briefly, total DNA was PCR-amplified using primers targeting the 16S V3 and V4 region (Illumina) (Klindworth et al. 2013 (link)) under the following conditions: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s and a final extension of 72°C for 10 min. The resulting 16S rDNA amplicons were run on a 1% agarose gel, size-selected at 450–500 bp and gel-purified using QIAquick Gel Purification kit (Qiagen). A second round of PCR was performed to add Nextera XT indices (Illumina) to purified amplicons. Indexed PCR products were purified with Ampure XP beads (Beckman Coulter, Brea, CA, USA) and quantified with Qubit dsDNA system (ThermoFisher Scientific). Samples were then normalized and pooled into sequencing libraries at 20 nM for oral wash and fecal samples and 1 nM for urine samples, then validated on a Bioanalyzer DNA 1000 chip (Agilent) and sequenced on the Illumina MiSeq with a V3 reagent kit at the Case Western Reserve University Genomics Core Facility.
7
Amplification and Sequencing of HIV cDNA
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A first round of amplification of the cDNA product was with a set of forward and reverse primers with an NGS adapter sequence at their 5′ ends and sequence complementary to the cDNA product at their 3′ ends. This set of primers included the common forward primer, HIV_NGS_F1697, whose 3′ sequence is complementary to the 3′ end of the cDNA. The reverse primer used for set I (‘long RNA: long cDNA’) was HIV_NGS_R1695, and for sets 2 and 3 (‘long RNA: short cDNA’ and ‘short RNA: short cDNA’, respectively) it was HIV_NGS_R1696. PCR products were then purified using Zymo-SpinTM I column (Zymo Research) with buffers from the PCR clean-up kit (Roche). Subsequently, a limited cycle PCR using Nextera XT indices (Illumina) was performed to incorporate sequencing adaptors and dual-index barcodes to each PCR product. Products were then purified with Ampure XP beads (Beckman Coulter), quantified using the Qubit fluorimeter (Thermo Fisher) and pooled at an equimolar concentration. The mix of 80 samples was sequenced on a MiSEq (Illumina) using a MiSeq Reagent Nano Kit (300 cycles) with V2 chemistry (Illumina).
8
Bacterial 16S rRNA Sequencing of Microbiome
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Microbial profiles were determined by sequencing a ~460 bp region including the V3 and V4 variable regions of bacterial 16s rRNA genes (primers in Supplementary Table 1 ) using a high-fidelity polymerase (NEG Q5, New England Biolabs). For longitudinal GF experiments, 2 μL of DNA per joint was used as PCR input. For cecal experiments approximately 30 ng of DNA was used as input from each sample. Illumina Nextera XT indices were attached, pooled in equimolar amounts, and sequenced on an Illumina miSeq sequencer using a 250 bp paired-end sequencing protocol by the Clinical Genomics Center at OMRF. Four cecal samples (2 old chow, 1 old HFD, 1 young chow + DMM) and 1 cartilage sample (old HFD) were excluded from analysis due to failed PCR amplification and/or 16S sequencing. No GF cartilage samples were excluded from analysis.
9
ATAC-seq Library Preparation Protocol
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ATAC-seq libraries were prepared using the Omni ATAC-seq protocol as described in Corces et al. (2017) (link). Adherent macrophages were washed once with PBS and lifted off of the plate with EDTA for 5 min. EDTA was quenched with RPMI, and library preparation was performed on 50,000 cells. 3.75 μL of Illumina Nextera XT indices were used in PCR and qPCR.
After performing the initial 5 cycles of PCR, 5% of the PCR reaction was used in qPCR to determine how many additional cycles were required. 4–7 cycles were determined to be sufficient for the final amplification. A 2-sided bead cleanup with AMPure XP beads was performed (0.5X, then 1.3X). Libraries were quantified using Qubit (dsDNA HS Assay) and the KAPA Library Quantification kit. Libraries from each timepoint were pooled to a concentration of 8 nM or 10 nM for each biological replicate, and 75-bp paired-end reads were sequenced on an Illumina NextSeq 500 using a High Output Kit.
After performing the initial 5 cycles of PCR, 5% of the PCR reaction was used in qPCR to determine how many additional cycles were required. 4–7 cycles were determined to be sufficient for the final amplification. A 2-sided bead cleanup with AMPure XP beads was performed (0.5X, then 1.3X). Libraries were quantified using Qubit (dsDNA HS Assay) and the KAPA Library Quantification kit. Libraries from each timepoint were pooled to a concentration of 8 nM or 10 nM for each biological replicate, and 75-bp paired-end reads were sequenced on an Illumina NextSeq 500 using a High Output Kit.
10
16S rRNA Gene Amplification and Sequencing
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For each sample, the V1–V2 region of the 16S rRNA gene was amplified using the primer set 27Fmod (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTTGATYMTGGCTCAG-3′) and 338R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTGCCTCCCGTAGGAGT-3′). The 16S rRNA gene was amplified in 25 μL reaction volumes containing 2.5 μL template DNA, 12.5 μL of 2 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA), and 5 μL of each primer (1.0 μM). Reactions were held at 95 °C for 3 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and then a final extension at 72 °C for 5 min. Amplified PCR products were purified using the AMPure XP system (Beckman Coulter, Brea, CA) and eluted in 52.5 μL Tris–HCL in DEPC-treated water. Then, 2.5 μL of the purified PCR product was used as a template for the second-round PCR reaction to attach Nextera XT indices and sequencing adapters (Illumina, San Diego, CA). PCR was performed at 95 °C for 3 min, followed by eight cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and then a final extension at 72 °C for 5 min. DNA sequencing was performed with the iSeq 100 system (Illumina) by 151-bp paired-end reads.
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