Fecal samples were collected and stored at −80°C prior to analysis. The standard PowerSoil kit protocol was used to extract bacterial genomic DNA. Briefly, the frozen samples were thawed on ice and then pulverized with a pestle and mortar in liquid nitrogen, we added MoBio lysis buffer to these fecal samples and then mixed them, and after centrifuging, we placed the obtained supernatant into MoBio garnet-bead tubes containing MoBio buffer. Extracted V3–V5 regions of the 16S rRNA gene from these fecal samples were amplified by polymerase chain reaction with bar-coded universal primers containing linker sequences for pyrosequencing.21 (link) The 454 sequencing system (Hoffman-La Roche, Basel, Switzerland) was used.
454 sequencing system
Manufactured by Roche
Sourced in United States
The Roche 454 sequencing system is a next-generation DNA sequencing platform that utilizes pyrosequencing technology to determine the nucleotide sequence of genetic samples. The system is designed to generate high-throughput sequencing data quickly and efficiently.
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Lab products found in correlation
7 protocols using 454 sequencing system
1
Fecal Microbiome Profiling Protocol
2
Soil Bacterial Diversity Assessment
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The quantity and quality of the pooled DNA extracted from the soil was assessed on 1% agarose gel. Bacterial diversity was assessed using the 16S rRNA gene, which was amplified using primers 968F (Nielsen et al., 1999) and 1378R. PCR was performed on a final volume of 50 µL, containing 1X enzyme buffer, 5 mM MgCl 2 , 10 mM dNTPs, 0.1 M each primer, and 2U Taq DNA polymerase. The amplification was performed in a thermal cycler, with an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 40 s, and a final extension at 72°C for 10 min. The PCR products were assessed using 1% agarose gel electrophoresis.
The PCR products were purified using Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK), following the manufacturer protocol. The purity and quantity of the amplicons were checked on 1% agarose gel. The PCR products of each treatment were mixed proportionally and were sequenced by a 454 Sequencing System (Roche Applied Science, Branford, CT, USA), using the pyrosequencing method.
The PCR products were purified using Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK), following the manufacturer protocol. The purity and quantity of the amplicons were checked on 1% agarose gel. The PCR products of each treatment were mixed proportionally and were sequenced by a 454 Sequencing System (Roche Applied Science, Branford, CT, USA), using the pyrosequencing method.
3
Microbial Community Analysis of Biofilms
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Total DNA was extracted from 1 mL of homogenate using a PowerBiofilm DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) in accordance with the manufacturer’s instructions.
To identify the microbial constituents of the biofilms, 16S rRNA bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Roche 454 sequencing system as previously described [24 ]. Amplicons originating from the V1–V3 region (27F–5' GAG TTT GAT CNT GGC TCA G 3' to 519R 5' GTN TTA CNG CGG CKG CTG 3' , numbered in relation to the E. coli 16S rRNA gene), were sequenced.
To identify the microbial constituents of the biofilms, 16S rRNA bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Roche 454 sequencing system as previously described [24 ]. Amplicons originating from the V1–V3 region (27F–
4
Fecal Microbiome Analysis in Mice
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Fecal samples were collected from the mice, and then immediately frozen and stored under −80 °C before analysis. We used the mortar and pestle to pulverize the fecal samples, and used the standard power soil kit protocol to extract the bacterial genomic DNA. Briefly, the fecal samples were thawed on the ice. Then, we added the MoBio lysis buffer into the fecal samples and conducted vortex mixing. The obtained fecal suspensions were centrifuged. Finally, we put the obtained supernatant into the MoBio Garnet bead tubes containing MoBio buffer. We used the Roche 454 sequencing system to extract the V3-V5 regions of 16s rRNA gene sequences from the fecal samples, and the obtained gene sequences were PCR-amplified with barcoded universal primers.
We used Mothur (Version 1.31.2,http://www.mothur.org/ ) to quality-filter the obtained raw gene sequences to collect unique reads. Sequences with <200 bp or >1000 bp, as well as sequences with any barcode mismatches, ambiguous bases, homopolymer runs exceeding six bases and primer mismatches were excluded. The remained sequences were assigned to operational taxonomic units (OTUs) with >=97% pairwise sequence identity. The Ribosomal Database Project (RDP) reference database was used to taxonomically classify the obtained OTUs.
We used Mothur (Version 1.31.2,
5
Bacterial DNA Extraction and Sequencing
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The DNA swabs (n = 30) were mixed with sterile PBS as described above and the samples were kept in −80 °C until DNA extraction. Biopsy samples (one caruncular and one intercaruncular sample per cow; n = 5 cows, 10 biopsies) were thawed and separately homogenized according to the extraction kit instructions. To avoid bias toward extraction of DNA from Gram negative bacteria, 2 mg of lysozyme (Thermo Fisher Scientific, Cat. No. 89833) were added to each sample and incubated at 37 °C for 1 h. Total DNA was extracted from swabs and biopsy samples using AccuPrep® Genomic DNA Extraction Kit (BIONEER, Republic of Korea, Cat. No. k-3032) following the manufacturer’s protocol.
Total DNA was used for 16S-rDNA bacterial tag encoded FLX amplicon pyrosequencing using the Roche 454 sequencing system as described previously [36 (link)]. The primers used for amplification prior to sequencing were 27F (5′ GAG TTT GAT CNT GGC TCA G 3′) and 519R (5′ GTN TTA CNG CGG CKG CTG 3′), resulting in amplicons containing the V1–V3 region of the 16S-rRNA bacterial genes.
Total DNA was used for 16S-rDNA bacterial tag encoded FLX amplicon pyrosequencing using the Roche 454 sequencing system as described previously [36 (link)]. The primers used for amplification prior to sequencing were 27F (5′ GAG TTT GAT CNT GGC TCA G 3′) and 519R (5′ GTN TTA CNG CGG CKG CTG 3′), resulting in amplicons containing the V1–V3 region of the 16S-rRNA bacterial genes.
6
Bacterial 16S rRNA Gene Amplification and Sequencing
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PCR amplification was done by using primers targeting the V1 to V3 regions of bacterial 16S rRNA gene with bacterial gDNA. For bacterial amplification, barcoded primers of 9F (5′-CCTATCCC-CTGTGTGCCTTGGCAGTC-TCAG-AC-AGAGTTTGATCMTGGCTCAG -3′; underlined sequence indicates the target region primer) and 541R (5′-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-ATTACCGCGGCTGCTGG -3′; ‘X’ presents the unique barcode for each subject) (http://oklbb.ezbiocloud.net/content/1001 ) as previous study were shown. The sequencing was performed at Chunlab Inc. (Seoul, Korea) with GS Junior Sequencing system, the modified laboratory benchtop form of 454 sequencing systems (Roche, Branford, CT, USA) as stated in the manufacturer’s directions.
7
Gut Microbiome Profiling via 16S rRNA
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The mucosal and gastric fluid samples from 4 subjects were subjected to pyrosequencing. Total genomic DNA was separated using a commercial kit (iNtRON Biotechnology, Seongnam, Korea). PCR amplification was done by taking primers targeting the V1 to V3 regions of the 16S rRNA gene with extracted DNA. For bacterial amplification, barcoded primer of 9F (5’-CCTATCCCC-TGTGTGCCTTGGCAGTC-TCAG-AC-AGAGTTTGATCMTGGCTCAG - 3’; underlined sequence indicates the target region primer) and 541R (5’-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-ATTACCGCGGCTGCTGG -3’; ‘X’ presents the unique barcode for each subject) (http://oklbb.ezbiocloud.net/content/1001 ) as previous study shows. The sequencing was performed at Chunlab (Seoul, Korea) with GS Junior Sequencing system, the modified laboratory benchtop form of 454 sequencing systems (Roche, Branford, CT, USA) as stated in the manufacturer’s directions.
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