We used the PBAT capture protocol combining PBAT and hybridization with an RNA probe library (capture), as described in detail in Meir Z. et al. Nature Genetics 2020, 10.1038/s41588-020-0645-y. Bisulfite conversion was performed with the EZ DNA Methylation-Lightning Kit (Zymo Research cat#D5031) following the manufacturer’s instructions. Converted DNA samples were subjected to an End repair reaction containing End repair mix and buffer (NEB cat#E6050) and 0.2–150 ng DNA. DNA was purified using 2.5× SPRI Agencourt AMPure XP beads (Beckman Coulter cat#A63881). The eluted product was next subjected to an A-tail reaction including 10 mM dATPs and Klenow Fragment 3′→5′ exo- (NEB cat#M0212). DNA was purified with 2.5× SPRI beads. The clean DNA was tagged with an index oligo adapter in a ligation reaction using the Quick ligase Kit (NEB cat#M2200). Tagged products were then cleaned using 1.3× SPRI beads, and amplified for library preparation with 14 PCR cycles using the KAPA HiFi HotStart Ready Mix kit (Kapa Biosystems cat#KK2601), following the manufacturer’s protocol. The reaction mix was then cleaned with 0.7× beads. Final libraries were pooled and sequenced on an Illumina NextSeq system using the 150-bp high-output sequencing kit.
Kapa hifi hotstart readymix kit
Manufactured by Roche
Sourced in United States
The KAPA HiFi HotStart ReadyMix kit is a pre-mixed solution designed for high-fidelity DNA amplification. It contains all the necessary reagents, including a high-performance DNA polymerase, dNTPs, and buffer components, in a ready-to-use format.
Automatically generated - may contain errors
Lab products found in correlation
Miseq sequencing system, by Illumina (3 mentions)
Miseq system, by Illumina (3 mentions)
Nextera barcode, by Illumina (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
16s metagenomic sequencing library preparation protocol, by Illumina (2 mentions)
Agencourt ampure xp system, by Beckman Coulter (2 mentions)
Quick ligase kit, by New England Biolabs (1 mentions)
Nextseq system, by Illumina (1 mentions)
Ez dna methylation lightning kit, by Zymo Research (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Ampure xp kit, by Beckman Coulter (1 mentions)
Agencourt ampure xp kit, by Beckman Coulter (1 mentions)
Miseq apparatus, by Illumina (1 mentions)
Miseq reagent kit v3 600 cycle, by Illumina (1 mentions)
16s metagenomics library preparation protocol, by Illumina (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Fastdna spin kit, by MP Biomedicals (1 mentions)
Genomic dna extraction kit, by Qiagen (1 mentions)
10 protocols using kapa hifi hotstart readymix kit
1
PBAT Capture Protocol for DNA Methylation
2
Illumina MiSeq 16S rRNA Sequencing
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The 16S rRNA gene ranged from V3 to V4 variable region was used as the target for bacterial community investigation by Illumina Miseq sequencing. PCR amplication primer was used according to Klindworth et al. [34 (link)] study and the protocal of library preparation guideline of Illumina. In general, PCR was performed by using KAPA HiFi HotStart ReadyMix kit (Kapa, Biosystems). Each PCR reaction contained genomic DNA 2.5 μl, forward and reverse primers 5 μl respectively, and KAPA mixture 12.5 μl. The amplication procedure was based on our previous study, and then the products were purified with AMPure XP magnetic beads (Beckman, Coulter), quantified using Qubit fluorometer (Invitrogen, Life Technologies). The secondary PCR amplication was performed to add the Illumina Nextera barcodes, using i5 and i7 primers following the manufacturer’s instruction, and then the purification process was executed again to remove nontarget fragments. Finally, the amplicons were normalized, pooled and sequencing was conducted using Illumina Miseq sequencing system (Illumina, SanDiego, USA).
3
16S rRNA Gene Amplification and Sequencing
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The V4 region of 16S rRNA gene was amplified by PCR with forward and reverse primers according to Kozich et al. (28 (link)), containing Illumina (Illumina Inc., San Diego, CA, USA) adapter sequences and unique dual indexes used to tag each PCR product, according to the 16S-protocol provided by Illumina. Primer sequences can be found in Table 1 . Briefly, PCR was carried out in 25 μL reactions with 0.2 μM forward and reverse primers, with 12.5 ng template DNA and 12.5 μL of 2 × KAPA HiFi HotStart Ready Mix kit (KAPA Biosystems, Woburn, MA, USA). Thermal cycling consisted of initial denaturation at 95°C for 3 min followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by a final step of 72°C for 5 min. The amplicon products were purified with Agencourt AmPureXP Kit (Beckman Coulter, Miami, FL, USA). Next, a second PCR was thereafter performed to attach Illumina adapters and unique dual indexes to each sample, followed by a clean-up step with AmPureXP Kit (Beckman Coulter). PCR amplicons were visualized using 0.1% agarose gel electrophoresis to verify the size of the amplicon. Negative extraction controls did not produce visible bands.
4
Characterization of Microbial Communities via 16S rRNA Sequencing
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The concentration of purified DNA was determined spectrophotometrically and DNA samples diluted to 5 ng/mL were used as a template in PCR with forward primer 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-MID-GT-CCTACGGGNGGCWGCAG-3′ and reverse primer 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-MID-GT-GACTACHVGGGTATCTAATCC-3′. MIDs represent different sequences of 5, 6, 7, or 9 nucleotides in length, which were used to identify individual samples in the sequencing run. PCR amplification was performed using a KAPA HiFi Hot Start Ready Mix kit (Kapa Biosystems, Woburn, MA, USA) and the resulting PCR products were purified using AMPure beads. Sequencing was performed using MiSeq Reagent Kit v3 (600 cycle) and MiSeq apparatus according to the manufacturer’s instructions (Illumina, San Diego, CA, USA), as described previously [6 (link)]. The fastq files were imported into QIIME software [14 (link)]. Forward and reverse sequences were joined, and chimeric sequences were predicted by the slayer algorithm and excluded from downstream analyses. The non-chimeric sequences were classified by RDP Seqmatch with an operational taxonomic units (OTU) discrimination level set to 97%. Principal coordinate analysis (PCoA) implemented in QIIME was used for data visualisation.
5
Illumina 16S Metagenomics Library Preparation
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The standard Illumina 16S metagenomics library preparation protocol was used (available at: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf ). All amplification and sequencing steps were carried out at University Core DNA Services, Sequencing and Genetic Analysis Lab (University of Calgary, AB, Canada). The 16S Amplicon PCR forward primer (5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and reverse primer (5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) were used to amplify the V3 and V4 regions of the 16S rRNA gene. Illumina sequencing adapters and dual-index barcodes were added to the amplicon target to allow for library pooling prior to sequencing. Briefly, 16S rRNA gene amplicons were generated using a KAPA HiFi HotStart ReadyMix Kit (Kapa Biosystems) with the following PCR conditions: a 3 min initial denaturation at 95 °C followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension of 5 min at 72 °C. Amplicon were then purified with Agencourt AMPure XP beads (Beckman Coulter Inc., ON, Canada), and sequenced on an Illumina MiSeq system (Illumina Inc., Victoria, BC, Canada) using the 2 × 300 bp paired-end sequencing kit. Negative controls were included in triplicate during amplification and sequencing.
6
16S rDNA Profiling of Kimchi Microbiome
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The bacterial community analysis was performed by CJ-Bioscience (Seoul, Korea) using 16S rDNA pyrosequencing with total DNA extracted from kimchi. Total DNA extraction was carried out following the manufacturer's instructions using the Fast DNA Spin kit (MP Biomedicals, CA, USA). A 16S rDNA sequencing library was prepared using the 16S metagenomic sequencing library preparation protocol (Illumina, San Diego, CA, USA). The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified and purified using the KAPA HiFi HotStart ReadyMix kit (KAPA Biosystems, Wilmington, MA, USA) and Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA).
Quality assessment and product size analyses were performed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. The amplified fragments were pooled, and sequencing was conducted on an Illumina MiSeq Sequencing System (Illumina, CA, USA) following the manufacturer's instructions. Raw reads were processed by initially checking the quality and filtering out low-quality reads (
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Quality assessment and product size analyses were performed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. The amplified fragments were pooled, and sequencing was conducted on an Illumina MiSeq Sequencing System (Illumina, CA, USA) following the manufacturer's instructions. Raw reads were processed by initially checking the quality and filtering out low-quality reads (
7
16S rDNA Pyrosequencing of Bacterial Communities
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Bacterial community analysis was performed using 16S rDNA pyrosequencing of genomic DNA isolated from the samples. A pyrosequencing library was generated using the 16S metagenomics sequencing library preparation protocol (Illumina MiSeq System, Illumina Inc., San Diego, CA, USA) by Theragen Inc. (Suwon, Korea). The V3 and V4 hypervariable regions were amplified using the KAPA HiFi HotStart ReadyMix Kit (KAPA Biosystems, Wilmington, MA, USA). The processing of raw reads began with a quality assessment, and reads with low quality (a score < Q25) were eliminated using Trimmomatic version 0.32. The paired-end sequence data were merged using fastq-mergepairs in VSEARCH version 2.13.4. Subsequently, redundant reads were identified and clustered with the unique reads. Taxonomic assignment was performed using the EzBioCloud 16S rRNA database, followed by a pairwise alignment. Chimeric reads were eliminated using reference-based chimeric detection with the UCHIME algorithm with a threshold set at <97% similarity. After the chimeric filtering step, the reads were compiled and cluster_fast command was utilized to generate additional OTUs. Data analysis was conducted using the EzBioCloud 16S-databased MTP provided by CJ Bioscience (Seoul, Korea) [15 (link)].
8
Profiling Kimchi Bacterial Diversity
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Genomic DNA was extracted from the kimchi samples using a genomic DNA extraction kit (Qiagen, Hilden, Germany). The bacterial communities were analyzed via pyrosequencing of the 16S rDNA. The samples were analyzed by Theragen (Suwon, Korea) using an Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA). A 16S rDNA-sequencing library was constructed using the 16S metagenomic sequencing library preparation protocol (Illumina). The V3 and V4 hypervariable regions of the 16S rDNA gene were amplified using the KAPA HiFi HotStart ReadyMix kit (KAPA Biosystems, Wilmington, MA, USA) and purified using the Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA). The prepared library sequences were run on the Miseq system (Illumina) with 2 × 300 bp paired-end reads. The reads were sorted using unique barcodes for PCR products, after which the barcode, linker, and primer sequences were removed from the original sequencing reads. Potential chimeric sequences were detected using the Bellerophon method. The number of operational taxonomic units (OTUs) were calculated from the pre-processed reads and determined by clustering the sequences using the QIIME software (v.1.8.0). Taxonomic abundance was counted with an RDP Classifier v1.1which was also used for normalization of microbial composition.
9
16S rRNA Gene Amplification and Sequencing
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The V3 to V4 variable region of the 16S rRNA gene was amplified using previously described primers [31 (link)]. Library preparation followed guidelines from Illumina. PCR was performed using the KAPA HiFi HotStart ReadyMix kit (Kapa, Biosystems). The amplification procedure has been previously described. PCR products were purified with AMPure XP magnetic beads (Beckman, Coulter) and quantified using a Qubit fluorometer (Invitrogen, Life Technologies). Secondary PCR amplification was performed to add Illumina Nextera barcodes and the products we purified to remove nontarget fragments. Amplicons were normalized, pooled, and sequencing using an Illumina Miseq system (Illumina, San Diego, USA) [28 ].
10
16S rRNA Amplicon Sequencing of Gut Microbiome
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16S rRNA V3-V4 hyper variable region sequencing (Clevergene, Bangalore, India) was performed on the faecal samples collected from the mice prior to the sacrifice. The sequencing employed KAPA HiFi HotStart Ready Mix kit and 341F and 785R as the primers. Sequencing libraries were created by running, an additional 8 cycles of DNA polymerase chain reaction (PCR) using Illumina bar-coded adapters. The sequencing data was generated by Illumina MiSeq software (USA). GREENGENES v.13.8-99 database, was employed to filter contigs and their organization into operational taxonomic units (OTU). The abundance of these OTU was estimated Fisher's exact test was used to determine the statistical significant differences in OTU abundance between samples (Schierová et al. 2020 (link); Peng et al. 2020 (link)).
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