Nine cDNA libraries were prepared using an Illumina RNA-Seq sample preparation kit (RNeasy Micro kit, Cat.#74004, Qiagen, China) using 10 μg of total RNA. The mRNA was isolated by polyA selection with oligo (dT) beads and fragmented using fragmentation buffer. Synthesis of cDNA, end repair, A-base addition, and ligation of the Illumina-indexed adaptors were then performed according to the Illumina protocol. Libraries were size-selected on 2% Low-Range Ultra Agarose (Bio-Rad Laboratories (Shanghai) Co., Ltd) for cDNA target fragments of 300–500 bp; this was followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. Following quantification by TBS380, paired-end libraries were sequenced using the Illumina HiSeq 2500 system (2×100 nt multiplex) at Shanghai Biotechnology Co., Ltd. (Shanghai, China). Data analysis and base calling were performed with the Illumina instrument software.
Phusion dna polymerase
Manufactured by Bio-Rad
Sourced in United States
Phusion DNA polymerase is a high-fidelity DNA polymerase enzyme used for DNA amplification in molecular biology applications. It has proofreading activity and can generate long PCR products with high accuracy.
Automatically generated - may contain errors
Lab products found in correlation
T100 thermal cycler, by Bio-Rad (2 mentions)
Instrument software, by Illumina (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Low range ultra agarose, by Bio-Rad (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Hiseq x10 platform, by Illumina (1 mentions)
Tbs380 picogreen, by Thermo Fisher Scientific (1 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Certified low range ultra agarose, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
4 protocols using phusion dna polymerase
1
Illumina RNA-Seq Library Preparation
2
Transcriptome Library Construction and Sequencing
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Transcriptome library was constructed with 1 μg of total RNA with concentration ≥ 30 ng/μL, RIN > 6.5 and OD260/280 between 1.8 and 2.2. Messenger RNA was isolated using the polyA selection method with oligo (dT) beads. Subsequently, the isolated mRNA was fragmented, and then double-stranded cDNA was synthesized with random hexamer primers. Synthesized cDNA underwent end-repair, phosphorylation and the addition of an “A” base. Libraries were size-selected for cDNA fragments of approximately 300 bp using 2% Low Range Ultra Agarose. Subsequently, PCR amplification was performed using Phusion DNA polymerase (NEB) for 15 PCR cycles (T100 Thermal Cycler, BIO-RAD, Hercules, CA, USA). The quantification of the libraries was carried out using the TBS380 system. The NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) was used to perform sequencing, with a read length of 2 × 150 bp [42 (link)].
3
Illumina RNA Sequencing Protocol
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The head sample was shipped in dry ice to Major Biosystem Corporation, Taiwan for RNA extraction, cDNA library construction, and Illumina sequencing. Total RNA was extracted using TRIzol® reagent (Invitrogen, USA) according to the manufacturer's protocol. RNA purity was checked using a NanoDrop 2000 spectrophotometer. The RNA integrity was observed on 1% agarose gel electrophoresis and RNA integrity value was analyzed using Agilent 2100 Bioanalyzer. The sample was verified to have a RIN (RNA integrity number) value of 7.4. The RNA was used for the building of cDNA libraries using the Truseq™ RNA sample prep Kit (Illumina, San Diego, USA) as recommended by the manufacturer. The cDNA was amplified by PCR using Phusion DNA polymerase (NEB) for 15 PCR cycles and cDNA Library was obtained by gel extraction selected for target fragments on 2% Low Range Ultra Agarose (Certified Low Range Ultra Agarose, Bio-Rad). The library was then quantified using TBS380（Picogreen, Invitrogen, Carlsbad, CA, USA）followed by bridge PCR amplification using the cBot Cluster Generation System (Truseq PE Cluster Kit v3-cBot-HS, Illumina, San Diego, USA). At last, the cDNA library was sequenced on the Illumina Hiseq X10 platform which generated 2 × 150 bp of paired-end raw reads.
4
Cloning and Expression of Bacillus subtilis ansZP21 Gene
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The B. subtilis CH11 strain was grown in TSB medium for 24 h at 37 • C. The genomic DNA was extracted according to Montes et al. [25] . The ansZ gene without the signal peptide YccC (first 60 base pairs), denominated as ansZP21, was amplified by PCR from the extracted DNA of Bacillus sp. CH11 using the forward primer 5 -TTT CAT ATG CCA CAT TCT CC T GAA ACA AAA GAA TCC CC-3 and the reverse primer 5 -TGC CGG ATC CTC AAT ACT CAT TGA AAT AAG C-3 . The gene was cloned using the restriction enzymes NdeI and BamHI, whose recognition sequences are in bold in the primers detailed above. PCR was carried out using Phusion DNA polymerase (2 U µL -1 ); the reaction conditions were an initial denaturation at 98 • C for 30 s, followed by 35 cycles at 98 • C for 10 s, 58 • C for 30 s, 72 • C for 20 s, and a final extension at 72 • C for 5 min (T100 Thermal Cycler, Bio-Rad, Hercules, CA, USA). The PCR products were cloned into pET-15b using 1 U of T4 DNA ligase and transformed into Escherichia coli DH5α. Then, the plasmids were extracted using kit QIAprep ® Spin Miniprep Kit and sent for sequencing to confirm the correct cloning of the ansZP21 gene. The correct expression vector was transformed into Escherichia coli BL21(DE3)pLysS host cells.
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