Nebnext ultra dna library preparation kit

ChIP-seq was performed as described previously [(54 (link)) and (17)]. Briefly, keratinocytes cultured in 10-cm² dishes were fixed in 1% formaldehyde for 5 min, and fixation was quenched with the addition of glycine to 125 mM for an additional 5 min. Cells were harvested by scraping from plates and washed twice in 1× PBS before storage at −80°C. ChIP extracts were sonicated for 15 min in a Covaris sonicator. All ChIPs were performed using 500 μg of extract and 2 μg of antibody per sample (anti-MLL4 ). Thirty microliters of Protein G Dynabeads was used per ChIP. ChIP DNA was also used to make sequencing libraries using a NEBNext Ultra DNA library preparation kit for Illumina. Library quality was checked by Agilent BioAnalyzer 2100, and libraries were quantified using the Library Quant Kit for Illumina. Libraries were then sequenced using a NextSeq500 33 platform (75-bp, single-end reads). After sequencing, all data were demultiplexed from the raw reads using Illumina’s BCL2FASTQ from BaseSpace. Further ChIP-seq analysis is described below.

A Qsonica Q800R sonicator was used to fragment the metagenomic DNA to ∼500 to 700 bp. A metagenome library was constructed with 500 ng of fragmented DNA using a NEBNext Ultra DNA library preparation kit for Illumina according to the manufacturer’s instructions. Quality control and sequencing of the metagenomic libraries were conducted at the University of Utah High-Throughput Genomics Core Facility. Libraries were evaluated for quality on a Bioanalyzer DNA 1000 chip (Agilent Technologies), and then paired-end sequencing (2 × 125 bp) was performed on an Illumina HiSeq2500 platform with HiSeq (v4) chemistry. The library was multiplexed with one other library (from a second Lost City chimney sample, results from which are not reported here) on one Illumina lane, yielding 180 million read pairs (45 billion bases). Demultiplexing and conversion of the raw sequencing base-call data were performed through the CASAVA (v1.8) pipeline.

ChIP DNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and the DNA integrity was checked with 4200 TapeStation (Agilent Technologies). ChIP-Seq library preparation and sequencing reactions were conducted at Azenta US, Inc. (South Plainfield, NJ, USA). NEB NextUltra DNA Library Preparation kit was used following the manufacturer’s recommendations (Illumina). Briefly, the ChIP DNA was end repaired and adapters were ligated after adenylation of the 3’ends. Adapter-ligated DNA was size selected, followed by clean up, and limited cycle PCR enrichment. The ChIP library was validated using Agilent TapeStation and quantified using Qubit 2.0 Fluorometer as well as real time PCR (Applied Biosystems). The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions (Illumina). Sequencing was performed using a 2×150 Paired End (PE) configuration. Image analysis and base calling were conducted by the Control Software (NCS). Raw sequence data (.bcl files) generated from the Illumina instrument was converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.

DNA was extracted from peripheral blood using the PureGene Genomic DNA purification kit (Qiagen, Valencia, CA). Two micrograms of genomic DNA were fragmented by sonication to a peak sized at 250bp for each sample. NEBNext Ultra DNA Library Preparation Kit Illumina (Life Technology, Grand Island, NY) was used for DNA library preparation with custom ligation adapter according to the manufacturer protocol. AMPure XP beads (Danvers, MA, Beckman Coulter Genomics) were used for double size selection to achieve ~250 bp inserts. Each sample was then barcoded by eight cycles of PCR amplification after which three indexed samples were pooled into one capture reaction. Hybridization was performed using SureSelect Human All Exon V5 probe library (Agilent Technologies, Santa Clara, CA) and streptavidin-coated magnetic beads were added to the capture mixture for targeted isolation. The purified DNA was then amplified again by 11 PCR cycles. All captured libraries were quantified using the Agilent Bioanalyzer for normalization and underwent 2X100 bp paired-end sequencing in HiSeq 2500 (Illumina, San Diego, CA) using a high-throughput mode.

The hmeDIP protocol was adapted from Mohn et al. (2009) and Maunakea et al. (2010) (link). The E16 VZ was subdissected as described for the VZ RNA-seq analysis in the Supplemental Material. The antibodies used for immunoprecipitation were 5mC (Millipore) and 5hmC (Active motif). DNA libraries were generated using the NEBNext Ultra DNA library preparation kit for Illumina. Quality control was carried out with a Bioanalyzer (Agilent), and 50-bp single-end sequencing was performed with a HiSeq sequencer (Illumina) at LAFUGA.