Fresh leaf tissue was sampled from a wild individual of R. nobile growing on Mount Segrila, Tibet, China (29°37′5.87″ N, 94°38′59.84″E, 4,628 m) and immediately stored in liquid nitrogen before sending to Grandomics (Wuhan, China) for genomic sequencing. High molecular weight genomic DNA was prepared by the CTAB method then purified with a QIAGEN® Genomic kit (Cat. No. 13343, QIAGEN). To obtain Illumina short reads, DNA libraries with 500 bp inserts were constructed and sequenced using an Illumina HiSeq 4000 platform. In addition, high-molecular-weight DNA was prepared and used to construct PacBio SMRTbell libraries using a SMRTbell Express Template Prep Kit 2.0, following the manufacturers’ recommended protocols. The SMRTbell libraries were sequenced using a PacBio Sequel II system and consensus (HiFi) reads were generated using ccs software (https://github.com/pacificbiosciences/unanimity ). Hi-C (high-throughput chromosome conformation capture) sequencing was performed as follows: sampled DNA was cross-linked with 1% formaldehyde to capture interacting DNA segments, chromatin was digested with the Dpn II restriction enzyme, and libraries were constructed and sequenced using the Illumina HiSeq 4000 platform.
Express template prep kit 2
Manufactured by Pacific Biosciences
The Express Template Prep Kit 2.0 is a laboratory tool designed for preparing DNA samples for sequencing analysis. It provides a streamlined workflow for library preparation, enabling efficient and reliable generation of sequencing-ready templates.
Automatically generated - may contain errors
3 protocols using express template prep kit 2
1
Genomic Sequencing of Rhodiola nobile
2
Whole Genome Sequencing of Microbial Strains
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The microbial strains were cultivated in Yeast-Malt-Glucose (YMG) broth for 48 h at 30 °C to produce the cell mass. The genomic DNA of strains Babs14 and Osf17 was initially extracted using Invitrogen PureLink® Genomic DNA kit. The DNA quantity and quality were tested by the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). The DNA was purified further using the Quick-DNA Miniprep Plus kit by Zymo research at the sequencing center of the University of Oregon Genomics & Cell Characterization Core Facility (GC3F). The whole genome sequencing was performed there by Pacific Biosciences Sequel II technology (PacBio). The DNA was made into SMRTbell libraries using the Express Template Prep Kit 2.0 from PacBio according to the manufacturer’s protocol. Samples were pooled into a single multiplexed library and size was selected using Sage Sciences’ BluePippin, which uses the 0.75% DF Marker S1 High-Pass 6 kb–10 kb v3 run protocol and S1 marker. A size selection cutoff of 8000 (BPstart value) was used. The size selected SMRTbell library was annealed and bound according to the SMRT Link Set Up and sequenced on a Sequel II. Raw PacBio reads were converted to fasta format with Samtools Fasta (http://www.htslib.org/doc/samtools.html ) and then assembled with Flye 2.6 (https://github.com/fenderglass/Flye ) with parameters-plasmids-iterations 2-asm-coverage 120.
3
Whole Genome Sequencing of Microbial Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microbial strains were cultivated in Yeast-Malt-Glucose (YMG) broth for 48 h at 30°C to produce cell mass. The genomic DNA of the strains Babs14 and Osf17 was initially extracted using Invitrogen PureLink® Genomic DNA kit. The DNA quantity and quality were tested by the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scienti c). The DNA was puri ed further by using the Quick-DNA Miniprep Plus kit by Zymo research at the sequencing center of the University of Oregon Genomics & Cell Characterization Core Facility (GC3F) and the whole genome sequencing was done there by Paci c Biosciences Sequel II technology (PacBio)The DNA was made into SMRTbell libraries using the Express Template Prep Kit 2.0 from PacBio according to the manufacturer's protocol. Samples were pooled into a single multiplexed library and size selected using Sage Sciences' BluePippin using the 0.75% DF Marker S1 High-Pass 6 kb-10 kb v3 run protocol and S1 marker. A size selection cutoff of 8000 (BPstart value) was used. The size-selected SMRTbell library was annealed and bound according to the SMRT Link Set Up and sequenced on a Sequel II. Raw PacBio reads were converted to fasta format with Samtools Fasta (http://www.htslib.org/doc/samtools.html) and then assembled with Flye 2.6 (https://github.com/fenderglass/Flye) with parameters-plasmids-iterations 2-asm-coverage 120.
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