MeD-seq assays were performed as previously described [20 (link)]. Briefly, 20 µL genomic DNA (input 90 ng) from frozen liver tissues was digested with LpnPI (New England Biolabs), generating 32 bp fragments around the methylated recognition site containing a CpG. These short DNA fragments were further processed using the ThruPlex DNA–seq 96D kit (Rubicon Genomics, Ann Arbor, MI, USA). Stem–loop adapters were blunt-end ligated to repaired input DNA, then amplified to include dual-indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA, USA). Multiplexed samples were sequenced on Illumina NextSeq2000 systems for paired-end reads of 50 bp, according to the manufacturer’s instructions. The dual-indexed samples were de-multiplexed using bcl2fastq v2.20 software (Illumina, San Diego, CA, USA).
Nextseq2000 systems
Manufactured by Illumina
The NextSeq2000 system is a high-throughput sequencing instrument designed for a range of genomic applications. It utilizes sequencing-by-synthesis technology to generate DNA sequence data. The system is capable of processing multiple samples simultaneously and producing large amounts of sequencing data in a single run.
Automatically generated - may contain errors
2 protocols using nextseq2000 systems
1
Genome-wide CpG Methylation Profiling via MeD-seq
2
MeD-seq Assay for Methylome Profiling
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MeD-seq assays were essentially performed as previously described 51 . Briefly, 10 µl of genomic DNA (input 90 ng) from naive hES cells was digested with LpnPI (New England Biolabs) generating 32-bp fragments around the fully methylated recognition site containing a CpG. These short DNA fragments were further processed using the ThruPLEX DNA-seq 96D Kit (Rubicon Genomics). Stem-loop adaptors were blunt-end ligated to repaired input DNA and amplified to include dual-indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science). Multiplexed samples were sequenced on Illumina NextSeq 2000 systems for singleend reads of 50 bp according to the manufacturer's instructions. Dual-indexed samples were demultiplexed using bcl2fastq software (Illumina).
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