DNA was extracted from 10–20 seedlings grown under long day conditions, using DNeasy Qiagen kits. About 10 mg of genomic DNA was sonicated to a 4–6 kb size range using the E210 Covaris instrument (Covaris, Inc., USA). Libraries were prepared following Illumina’s protocol (Illumina Mate Pair library kit), starting with size-selected (approximately 5 kb) fragments, which were end-repaired, biotin labeled and circularized. Linear DNA was eliminated by digestion and circularized DNA was fragmented to 300–700 bp using the E210 Covaris. Biotinylated DNA junctions were purified using streptavidin, end-repaired and 3′-adenylated in order to ligate Illumina adapters. Junction fragments were PCR-amplified using Illumina adapter-specific primers and amplified fragments within the 350–650 bp size range were selected for sequencing. Each library was sequenced using 100 base-length read chemistry in a paired-end flow cell on the Illumina GAIIx (2 lanes) or HiSeq2000 (1 lane) (Illumina, USA).
Mate pair library kit
Manufactured by Illumina
The Mate Pair library kit is a laboratory tool designed for the preparation of DNA libraries for Next-Generation Sequencing (NGS) applications. The kit enables the creation of long-range genomic libraries by circularizing DNA fragments and then selectively enriching for the junction fragments. This approach provides valuable information about the organization and structure of genomes, which is useful for various genomic research applications.
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Lab products found in correlation
3 protocols using mate pair library kit
1
Long-Read Genome Sequencing Protocol
2
High-Quality Genomic DNA Extraction and Sequencing
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DNA was extracted from seedlings grown under long-day conditions, using DNeasy Qiagen kits. About 10 microgram of genomic DNA were sonicated separately to a 4–6 Kb size range using the E210 covaris instrument (Covaris, Inc., USA). Libraries were prepared following Illumina’s protocol (Illumina Mate Pair library kit). Briefly, fragments were end-repaired and biotin labeled. A size selection of fragments with length of interest (around 5 Kb) was performed. DNA were then circularized and linear, non-circularized DNA were eliminated by digestion. Circularized DNA were fragmented to 300-700-bp size range using covaris E210. Biotinylated DNA were purified, end-repaired, then 3′-adenylated, and Illumina adapters were added. DNA fragments were PCR-amplified using Illumina adapter-specific primers. Finally, the PCR amplified libraries (350–650 bp) were size-selected. Libraries were then quantified using a Qubit Fluorometer (Life technologies) and libraries profiles were evaluated using an Agilent 2100 bioanalyzer (Agilent Technologies, USA). Each library was sequenced using 100 base-length read chemistry in a paired-end flow cell on the Illumina GAIIx (2 lanes) or HiSeq2000 (1 lane) (Illumina, USA).
3
Identifying EMS-induced Mutations in Yeast
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Using 20 four-spored tetrads from a cross between an EMS-induced hit and SLJ6121, we identified the two progeny from each tetrad that were diploid (control) and the two progeny from each tetrad that were haploid (and therefore contained an ems hit). To ensure equal representation of colonies, each was individually grown, normalized by OD600, then mixed to achieve equal number of all 40 cells in the control and ems hit pools. Genomic libraries were prepared using the Illumina Mate Pair library kit and prepared for paired-end sequencing on the Illumina MiSeq as previously described (Birkeland et al. 2010) . Downstream sequence analysis was performed (Birkeland et al. 2010) . Reads were aligned to sacCer3 using bwa version 0.7.15-r1140 (Li and Durbin 2009) and single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms were identified using SAMtools version 1.5 (Li et al. 2009) . SNP and insertion/deletion polymorphisms were annotated using snpEff version 4.3 (Cingolani et al. 2012) . Coverage was calculated using BEDTools version 2.25.0 (Quinlan and Hall 2010) . Results are listed in Table S1.
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