fspei  (New England Biolabs)

Journal: Genome Research

Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

doi: 10.1101/gr.222885.117

Figure Lengend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

Techniques: Ligation, Fractionation, Amplification, Sequencing

Journal: Open Biology

Article Title: MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

doi: 10.1098/rsob.150130

Figure Lengend Snippet: Schematic overview of the procedure for MethylRAD library preparation. Genomic DNA is digested with the restriction enzyme FspEI, producing 32-bp fragments including four-base 3′ overhangs. Adaptors with compatible overhangs (NNNN) are ligated to each end of these fragments. Tag density can be adjusted using adaptors with selective overhangs (e.g. NNNG). The constructs are amplified and purified by gel extraction. Sample-specific barcodes are incorporated in each construct by PCR, and the products pooled for sequencing.

Article Snippet: MethylRAD library preparation and sequencing MethylRAD library preparation began with digestion of 1–200 ng genomic DNA in a 15 µl reaction containing 4 U FspEI (NEB) at 37°C for 4 h. Five µl of the digested product was run on a 1% agarose gel to verify digestion.

Techniques: Construct, Amplification, Purification, Gel Extraction, Polymerase Chain Reaction, Sequencing

Journal: Genome Research

Article Title: Novel features of telomere biology revealed by the absence of telomeric DNA methylation

doi: 10.1101/gr.202465.115

Figure Lengend Snippet: Methylation-dependent restriction enzyme analyses confirm the absence of telomeric DNA methylation. ( A ) Cartoon representing the sequence specificity of the restriction enzymes used to digest Arabidopsis genomic DNA. ( B ) Southern blot hybridizations of Arabidopsis genomic DNA digested with the restriction enzymes indicated in A . The upper panel shows equal amounts of undigested (Control) or digested (HpaII, FspEI, or McrBC) DNA samples hybridized with a telomeric probe. The four DNA samples were also digested with Tru9I prior to hybridization. The middle panel shows the hybridization of the same samples with a 180-bp centromeric repeat probe. The ethidium bromide staining of the samples is shown in the lower panel. The control DNA sample was run in the same gel as the rest of the samples, so that their corresponding hybridization signals were processed equally. ( C ) Bar plot representation of the telomeric and centromeric bottom band hybridization signals expressed as percentages of the control. C, H, F, and M represent Control, HpaII, FspEI, and McrBC, respectively.

Article Snippet: Equal amounts of genomic DNA (500 ng) were undigested or digested with the restriction enzymes HpaII, FspEI, or McrBC following the supplier's instructions (New England Biolabs).

Techniques: Methylation, DNA Methylation Assay, Sequencing, Southern Blot, Hybridization, Staining