DNA concentration was measured using the Qubit dsDNA HS Assay Kit and size examined with the High Sensitivity DNA Analysis Kit (Genomics Agilent) using Bioanalyzer 2100 (Genomics Agilent). DNA was denatured with 0.2 M NaOH and its concentration was adjusted to 16pM. Samples were sequenced with the MiSeq Reagent Kit v3 (Illumina) at 51 cycles per run and single end reads. A 12.5 pM PhiX v3 library (Illumina) was added for a positive control.
Phix v3 library
Manufactured by Illumina
The PhiX v3 library is a synthetic DNA control material used to assess the performance of Illumina sequencing systems. It provides a known DNA sequence that can be used to monitor and optimize key sequencing metrics, such as cluster density, base-calling accuracy, and sequencing quality.
Automatically generated - may contain errors
Lab products found in correlation
Kapa library quantification kits for platforms, by Illumina (2 mentions)
Novaseq 6000 s2 flow cell, by Illumina (2 mentions)
Accel ngs methyl seq dna library preparation kit, by Integrated DNA Technologies (2 mentions)
Ez 96 dna methylation kit, by Zymo Research (2 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Gel purification kit, by Qiagen (1 mentions)
Ht1 buffer, by Illumina (1 mentions)
2100 bioanalyzer dna 1000 chip, by Agilent Technologies (1 mentions)
Miseq v2 500 cycle kit cassette, by Illumina (1 mentions)
Fragment analyzer ce, by Agilent Technologies (1 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using phix v3 library
1
Quantification and Sequencing of DNA
2
Illumina MiSeq Library Preparation
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Pooled PCR products were gel purified using the Qiagen Gel Purification Kit (Qiagen, Frederick, Maryland, USA). Clean PCR products were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and samples were combined in equimolar amounts. Prior to submission for sequencing, libraries were quality checked using the 2100 Bioanalyzer DNA 1000 chip (Agilent Technologies, Santa Clara, CA). Pooled libraries were stored at −20 °C until they were shipped on dry ice to the California State University (North Ridge, CA) for sequencing. Library pools were size verified using the Fragment Analyzer CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using the Qubit High Sensitivity dsDNA kit (Life Technologies, Carlsbad, CA). After dilution to a final concentration of 1 nM and a 10% spike of PhiX V3 library (Illumina, San Diego CA), pools were denatured for 5 minutes in an equal volume of 0.1 N NaOH, then further diluted to 12 pM in Illumina's HT1 buffer. The denatured and PhiX-spiked 12 pM pool was loaded on an Illumina MiSeq V2 500 cycle kit cassette with 16S rRNA library sequencing primers and set for 250 base, paired-end reads.
3
Bisulfite Conversion and Methylation Sequencing
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Up to 75 ng of sheared genomic DNA was subjected to bisulfite conversion using the EZ-96 DNA Methylation Kit (Zymo Research), with liquid handling on a MicroLab STAR (Hamilton). Dual-indexed sequencing libraries were prepared using Accel-NGS Methyl-Seq DNA library preparation kits (Swift BioSciences) and custom liquid handling scripts executed on the Hamilton MicroLab STAR. Libraries were quantified using KAPA Library Quantification Kits for Illumina Platforms (Kapa Biosystems). Four uniquely dual-indexed libraries, along with the 10% PhiX v.3 library (Illumina), were pooled and clustered on an Illumina NovaSeq 6000 S2 flow cell followed by 150 bp, paired-end sequencing. Total read count and average sequencing depth (in read pairs), as well as percentage of CpGs, per sample, at 1× and 10×, are detailed in Supplementary Table 1 . Also listed are average methylation levels, per sample, at CpG, nonCpG and CC dinucleotides. Intriguingly, sorted neuron samples showed higher CpA methylation (approximately 10%) compared with other samples (approximately 1%).
4
Bisulfite Conversion and Methyl-Seq Library Prep
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Up to 75 ng of sheared gDNA was subjected to bisulfite conversion using the EZ-96 DNA Methylation Kit (Zymo Research; Irvine, CA), with liquid handling on a Hamilton MicroLab STAR (Hamilton; Reno, NV). Dual indexed sequencing libraries were prepared using Accel-NGS Methyl-Seq DNA library preparation kits (Swift BioSciences; Ann Arbor, MI) and custom liquid handling scripts executed on the Hamilton MicroLab STAR. Libraries were quantified using KAPA Library Quantification Kits for Illumina Platforms (Kapa Biosystems; Wilmington, MA). Four uniquely dual indexed libraries, along with 10% PhiX v3 library (Illumina; San Diego, CA), were pooled and clustered on a Illumina NovaSeq 6000 S2 flow cell followed by 150-bp paired-end sequencing.
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