Total genomic DNA was extracted from 45-day-old leaves of the reciprocal hybrids and their parent plants using CTAB method as described by Murray and Thompson.27 (link) At this stage, the differences in leaf phenotype and plant morphology among the four genotypes of tomato plants became evident. Three seedlings were pooled in each genotype sample for genomic DNA extraction. The quality of DNA was analysed using agarose gel electrophoresis. Five μg DNA was then randomly fragmented into small pieces with most of them 100–500 bp in length and purified by QIA quick PCR Purification Kit (Qiagen, Hilden, Germany). Four DNA libraries were constructed, namely the M, W, M × W and W × M DNA libraries. After DNA-end repair and 3’-dA overhang, DNA was ligated to an Illumina sequencing primer adaptor. The double-stranded DNA was subsequently denatured and immunoprecipitated using 5mC antibody. Real time quantitative PCR analysis was performed to validate the quality of immunoprecipitated DNA fragments. DNA with high quality was used for PCR amplification and bands between 200-300 bp were selected, and subsequently sequenced using the HiSeq 2000 system (SBS KIT-HS V3, Illumina).
Sequencing primer adaptor
Manufactured by Illumina
Sourced in United States
The Sequencing primer adaptor is a laboratory equipment used in DNA sequencing workflows. It serves as a connector between the DNA sample and the sequencing platform, enabling the initiation of the sequencing process. The adaptor provides the necessary sequences for the sequencing primers to bind and facilitate the sequencing reaction.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Genomic dna miniprep kit, by Corning (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 analyzer, by Agilent Technologies (1 mentions)
Paired end dna sample prep kit, by Illumina (1 mentions)
Biophotometer plus spectrophotometer, by Eppendorf (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
2 protocols using sequencing primer adaptor
1
Genomic DNA Extraction and Sequencing
2
DNA Extraction, Fragmentation, and Sequencing
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Total genomic DNA was extracted using a Genomic DNA Miniprep Kit (Axygen) following the manufacturer’s instructions. The DNA quality was then evaluated by agarose gel electrophoresis and a BioPhotometer Plus spectrophotometer (Eppendorf, Germany).
Subsequently, the two samples were sonicated to produce DNA fragments ranging from 100 to 500 bp. After end repairing, phosphorylating and A-tailing with Paired-End DNA Sample Prep kit (Illumina, USA), the DNA was ligated to an Illumina sequencing primer adaptor. Then the fragments were used for methylated DNA immunoprecipitation (MeDIP) enrichment using a Magnetic Methylated DNA Immunoprecipitation kit (Diagenod, Belgium) following the manufacturer’s recommendations and the qualifying DNA was used for PCR amplification. Then bands between 220 and 320 bp were excised from the gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). The products were quantified with a Quant-iT™ dsDNA HS Assay Kit (Invitrogen, USA) using an Agilent 2100 Analyzer (Agilent Technologies, USA). Following qPCR qualification, DNA libraries were sequenced on the Illumina HiSeq 2000 (Illumina, CA, USA) to generate paired-end 50-bp reads by the Beijing Genomics Institute (BGI, China).
Subsequently, the two samples were sonicated to produce DNA fragments ranging from 100 to 500 bp. After end repairing, phosphorylating and A-tailing with Paired-End DNA Sample Prep kit (Illumina, USA), the DNA was ligated to an Illumina sequencing primer adaptor. Then the fragments were used for methylated DNA immunoprecipitation (MeDIP) enrichment using a Magnetic Methylated DNA Immunoprecipitation kit (Diagenod, Belgium) following the manufacturer’s recommendations and the qualifying DNA was used for PCR amplification. Then bands between 220 and 320 bp were excised from the gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). The products were quantified with a Quant-iT™ dsDNA HS Assay Kit (Invitrogen, USA) using an Agilent 2100 Analyzer (Agilent Technologies, USA). Following qPCR qualification, DNA libraries were sequenced on the Illumina HiSeq 2000 (Illumina, CA, USA) to generate paired-end 50-bp reads by the Beijing Genomics Institute (BGI, China).
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