Lower respiratory tract samples (pellets of BALF) were collected on 1, 3, 7, 14, and 28 dpi. There were 5/6 mice in each group. Every sample was analyzed independently. Total genomic DNA was extracted using the CTAB/SDS method, and quantified by electrophoresing through 1% agarose gels. The DNA samples were diluted to 1 ng/μl using sterile water, and the 16S rRNA genes were amplified using barcoded primers (Phusion® High-Fidelity PCR Master Mix, NEB, United States) specific for the V4 region (515F-806R) (Caporaso et al., 2012 (link)) and the Phusion® High-Fidelity PCR Master Mix (NEB, United States).
Phusion high fidelity pcr master mix
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, China, Germany
Phusion® High-Fidelity PCR Master Mix is a ready-to-use solution for high-fidelity DNA amplification. It contains Phusion® DNA Polymerase, dNTPs, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
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Agilent bioanalyzer 2100 system, by Agilent Technologies (90 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (78 mentions)
Hiseq 2500 platform, by Illumina (69 mentions)
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Miseq platform, by Illumina (31 mentions)
Qiaquick gel extraction kit, by Qiagen (26 mentions)
Ion plus fragment library kit 48 rxns, by Thermo Fisher Scientific (23 mentions)
Nebnext ultra dna library prep kit, by Illumina (19 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (18 mentions)
Hiseq platform, by Illumina (17 mentions)
Qiaamp dna stool mini kit, by Qiagen (16 mentions)
Novaseq 6000, by Illumina (14 mentions)
Novaseq 6000 platform, by Illumina (13 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (12 mentions)
Truseq dna pcr free library preparation kit, by Illumina (11 mentions)
Axyprep dna gel extraction kit, by Corning (10 mentions)
Hiseq 2500, by Illumina (10 mentions)
599 protocols using phusion high fidelity pcr master mix
1
Murine Lung Microbiome Profiling
2
Mammalian Expression Plasmids for PrPC
3
Targeted Gene Editing Efficiency Analysis
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At different time points after TALEN transfection, cells were harvested, washed once in PBS and subject to genomic DNA extraction by Mag‐Bind Blood & Tissue DNA HDQ kits (Omega Bio‐Tek).
For indel analysis, PCR amplifications spanning TRAC or B2M targets were performed using primers described in the Supplemental Materials. 1 μg genomic DNA was used per reaction in a 50 μL reaction with Phusion High‐Fidelity PCR Master Mix (NEB). The PCR condition was set to 1 cycle of 30 s at 98 °C; 30 cycles of 10 s at 98 °C, 30 s at 60 °C, 30 s at 72 °C; 1 cycle of 5 min at 72 °C; hold at 4 °C. The PCR product was then purified with Omega NGS beads (1 : 1.2 ratio) and eluted into 30 μL of 10 mm Tris buffer pH7.4. The second PCR which incorporates NGS indices to the sample was then performed on the purified product from the first PCR. 15 μL of the first PCR product was set in a 50 μL reaction with Phusion High‐Fidelity PCR Master Mix (NEB). The PCR condition was set to 1 cycle of 30 s at 98 °C; 8 cycles of 10 s at 98 °C, 30 s at 62 °C, 30 s at 72 °C; 1 cycle of 5 min at 72 °C; hold at 4 °C. Purified PCR products were sequenced on MiSeq (Illumina, San Diego, CA, USA) on a 2 x 250 Nano‐V2 cartridge. At least 150 000 sequences were obtained per PCR product, and sequences were analyzed for the presence of site‐specific indels.
For indel analysis, PCR amplifications spanning TRAC or B2M targets were performed using primers described in the Supplemental Materials. 1 μg genomic DNA was used per reaction in a 50 μL reaction with Phusion High‐Fidelity PCR Master Mix (NEB). The PCR condition was set to 1 cycle of 30 s at 98 °C; 30 cycles of 10 s at 98 °C, 30 s at 60 °C, 30 s at 72 °C; 1 cycle of 5 min at 72 °C; hold at 4 °C. The PCR product was then purified with Omega NGS beads (1 : 1.2 ratio) and eluted into 30 μL of 10 m
4
Genomic DNA Amplification and Sequencing
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100 μg genomic DNA was used per reaction in a 50 μL reaction with Phusion High-Fidelity PCR Master Mix (NEB). The PCR condition was set to 1 cycle of 30 s at 98°C; 30 cycles of 10 s at 98°C, 30s at 60°C, 30 s at 72°C; 1 cycle of 5 min at 72°C; hold at 4°C. The PCR product was then purified with Omega NGS beads (1:1.2 ratio) and eluted into 30 μL of 10 mM Tris buffer pH7.4. The second PCR which incorporates NGS indices was then performed on the purified product from the first PCR. 15 ul of the first PCR product were set in a 50 μL reaction with Phusion High-Fidelity PCR Master Mix (NEB). The PCR condition was set to 1 cycle of 30 s at 98°C; 8 cycles of 10 s at 98°C, 30 s at 62°C, 30s at 72°C; 1 cycle of 5 min at 72°C; hold at 4°C. Purified PCR products were sequenced on MiSeq (Illumina) on a 2 × 250 nano V2 cartridge.
5
Identifying CYP Candidate Genes in T. wilfordii
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CYPs were obtained from the PFAM database and annotated as cytochrome P450 (PF00067). A heatmap of gene expression was generated using MultiExperiment Viewer (MeV, version 4.9.0) and Java 8 software. The RPKM reads of the root periderm, root phloem, root xylem, stem vascular bundle, stem periderm, and leaf originated from previous tissue transcriptomes (NCBI SRA number SRP199495, Supplementary Fig. 20 )16 (link), and the data were normalized and processed by hierarchical cluster analysis with Pearson correlation. CYPs with gene expression profiles similar to those of TwTPS7(v2), TwTPS27(v2), and CYP728B70 were selected as candidate CYPs for further analysis. The candidate CYPs were amplified using 2×Phusion High-Fidelity PCR Master Mix (New England Biolabs, USA) and cDNA of T. wilfordii root as the template, which was consistent with the transcriptome after complete sequencing (Supplementary Data 5 ).
6
Amplicon Library Preparation for Illumina Sequencing
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The V3-V4 PCR products generated were 1/20-diluted, 1 μl of the diluted amplicon was used in the second PCR step. The second PCR incorporated Illumina flow-cell linkers and 8-bp dual-index barcodes to the amplicons. This was done using primers with Illumina flow cell linkers + 8-bp index barcodes + Illumina 5′ sequencing adapters. Primer sequences are shown in Supplementary Table S1 . PCR was performed using 10 μl 2× Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.4 μM of each primer and 1 μl of the diluted amplicon. PCR conditions involved an initial denaturation at 98°C for 30 s, 10 cycles consisting of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C for 15 s, and final extension at 72°C for 10 min.
After PCR amplification, amplicons were pooled in equal volume, the amplicon library was purified using QIAgen PCR Purification kit (QIAgen, Hilden, Germany). Quality of the library was assessed on Agilent Bioanalyser 2100 system (Agilent Technologies Inc., Santa Clara, CA, United States). The library was sequenced on an Illumina HiSeq2500 system (Illumina, San Diego, CA, United States) and 250 bp paired-end reads were generated.
After PCR amplification, amplicons were pooled in equal volume, the amplicon library was purified using QIAgen PCR Purification kit (QIAgen, Hilden, Germany). Quality of the library was assessed on Agilent Bioanalyser 2100 system (Agilent Technologies Inc., Santa Clara, CA, United States). The library was sequenced on an Illumina HiSeq2500 system (Illumina, San Diego, CA, United States) and 250 bp paired-end reads were generated.
7
PCR-Based Sanger Sequence Validation
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The polymerase chain reaction (PCR) for Sanger sequence validation was performed using the 2× Phusion High-Fidelity PCR master mix with HF buffer (NEB). Briefly, the PCR ran for 30 cycles with 1-min elongation at 72 °C. The PCR products were purified using Ampure XP beads following the guidelines of the manufacturer. The sizing of the amplicons was checked using Agilent’s Labonachip system. The Sanger sequencing of the products was performed by the LGTC and the sequences were analyzed using Sequence Scanner Sofware 2 (Applied Biosystems, CA USA).
8
Molecular Detection of SLCuV by PCR
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To detect and identify SLCuV, samples were tested by PCR. One microliter of total DNA from each sample (50 ng/µL) was used as template. The reaction mixture consisted of 0.5 µM forward and reverse primers (SqA2F and SqA1R; Table 1 ), 10 µL of 2×Phusion High-Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, MA, USA) in 20 µL of final reaction volume. The PCR reaction was carried out as follows: initial denaturation step (98 °C 30 s, one cycle), amplification step for 35 cycles (98 °C 10 s, 55 °C 30 s and 72 °C 30 s, for each cycle), and a final elongation step (72 °C, 5 min).
9
Genotyping GFP and GAPDH Genes
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To genotype the GFP and GAPDH genes, we inoculated single colonies in LB media and cultured them for 16 h at 34 °C. gfp and gapdh loci were amplified from 1 ul of 100 times diluted bacterial culture using 10 μl 2 × Phusion High-Fidelity PCR Master Mix (NEB), 7 μl water, and 1 μl of 10 μM primer(each) with thermocycling program of 98 °C for 2 min; (98 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min) × 30 cycles, 72 °C for 10 min. The primer sequences can be found in Supplementary Note 11 .
10
Bacterial Community Profiling with V3-V4 16S rDNA Amplicons
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For identification of bacterial communities, primers specific to the variable region V3 to V4 of the 16S rDNA of bacterial species were used, the forward primer was 341F and the reverse primer was 806R. Besides, the primers also contained Illumina 5′ sequencing adapters, so that the resultant V3-V4 amplicons were incorporated with Illumina 5′ sequencing adapters. Details of the primers are shown in Supplementary Table S1 . Amplification was performed using 10 μl 2× Phusion High-Fidelity PCR Master Mix (New England Biolabs, Beverly, MA, United States), 0.4 μM each primer, and 5 μl of genomic DNA. PCR conditions involved a denaturation at 98°C for 30 s, 30 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C for 15 s, and final extension at 72°C for 10 min.
Suitability of the V3 and V4 primers was evaluated in silico using the online tool arb-SILVA TestPrime (Quast et al., 2012 (link)) based on the 16S small subunit (ssur123) and non-redundant SILVA reference database (SILVA Ref NR). At one-mismatch-stringency, the V3-V4 specific primer pairs (excluding the Illumina 5′ sequencing adapters) showed 87% coverage for all bacterial phyla and considered suitable.
Suitability of the V3 and V4 primers was evaluated in silico using the online tool arb-SILVA TestPrime (Quast et al., 2012 (link)) based on the 16S small subunit (ssur123) and non-redundant SILVA reference database (SILVA Ref NR). At one-mismatch-stringency, the V3-V4 specific primer pairs (excluding the Illumina 5′ sequencing adapters) showed 87% coverage for all bacterial phyla and considered suitable.
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