Reduced Representation Bisulfite Sequencing (RRBS) was performed as described [7 (link)]. Briefly, DNA samples were incubated with MspI enzyme (NEB) in order to fragment DNA at CCGG positions to enrich for CpG regions. Bisulfite conversion was performed using the EZ DNA methylation Kit (Zymo Research) according to the manufacturer’s instructions. DNA was then PCR amplified and ligated to TruSeq (Illumina) sequencing adapters. Libraries were subjected to 75-bp single-end sequencing on a NextSeq 550 (Illumina). On average, 18.3 million reads/sample were aligned and 205304 CpGs had sufficient coverage in every sample.
Mspi enzyme
Manufactured by New England Biolabs
Sourced in United States
MspI is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-CCGG-3'. It is commonly used in molecular biology applications for DNA digestion and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ez dna methylation kit, by Zymo Research (2 mentions)
Imprint modification kit, by Merck Group (2 mentions)
Kapa uracil dna polymerase master mix, by Roche (2 mentions)
Nebnext library prep universal and index primers, by New England Biolabs (2 mentions)
Savant speedvac concentrator, by Thermo Fisher Scientific (2 mentions)
Zr duet dna rna miniprep kit, by Zymo Research (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Nextseq 550, by Illumina (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Pcr primers, by Thermo Fisher Scientific (1 mentions)
8 protocols using mspi enzyme
1
Reduced Representation Bisulfite Sequencing
2
Genome-wide DNA methylation profiling of PGCs
3
Reduced Representation Bisulfite Sequencing for DNA Methylation Profiling
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To allow comprehensive, unbiased profiling of DNA methylation, the RRBS method was followed to capture target regions for library preparation41 (link). 2.5 µg of genomic DNA was digested overnight with MspI enzyme (New England Biolabs, Ipswich, MA), using 20 units of enzyme per µg of DNA to ensure complete digestion. The size-selected targeted fragments were bisulfite converted using EZ DNA methylation kit (Zymo Research, Irvine, CA) and checked on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY). A double-stranded DNA library was produced using Illumina TruSeq DNA library preparation system (Illumina, San Diego, CA). These libraries were PCR amplified, normalised and qPCR was performed to confirm quality, and were then pooled by fluorometric quantitation readings in equimolar concentrations for two lanes of a Hiseq 2000 sequencer flow cell. We performed single-end sequencing with 51 bp read length. Read quality was examined by FastQC, using Phred score of 20 as a cutoff (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). All samples were screened and deemed of high quality for inclusion in subsequent analysis.
4
Promoter Methylation Analysis of Rat rDNA
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The rat rDNA fragments, recovered separately from total DNA or non-matrix-associated DNA and matrix-associated DNA after digestion of restriction enzymes (BamH1+Xho1), were subjected to digestion with HpaII or MspI enzyme (New England Biolabs) prior to qPCR analysis. HpaII/MspI restriction enzyme sensitivity assays were used to measure the methylation status of a promoter CpG site at position −145 (relative to the transcription initiation site) (40 (link)). A primer set, the forward primer: 5′-agcatggacttctgaggccgag-3′ and the reverse primer: 5′-cataaagctgccccagagag-3′, was used to amplify a fragment of 450 bp containing an HpaII/MspI restriction site that includes the −145-bp CpG site. CpG methylation at this site inhibits cleavage by HpaII and thus the level of resistance to cleavage can be used as a measure of CpG methylation. The R0 primer pair (Supplementary Table S2) amplifies a 200-bp fragment downstream of the transcription initiation site that was used for normalization between samples.
5
Bisulfite-based DNA Methylation Sequencing
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The 200 to 1,000 bp in length fragmented DNA samples by MspI enzyme (NEB, United States) were then subjected to bisulfite conversion for converting any unmethylated cytosine to Uracil by EZ DNA Methylation-Gold™ Kit (Zymo Research, United States). Further, the Accel-NGS Methyl-Seq DNA Library Kit (Swift, MI) was utilized for attaching adapters to single-stranded DNA fragments. Bead-based SPRI clean-ups were used to remove both oligonucleotides and small fragments, as well as changing enzymatic buffer composition. Finally, we performed the pair-end 2 × 150 bp sequencing on an Illumina Hiseq 4000 platform housed in the LC Sciences (Hangzhou, China).
6
Genome-wide DNA methylation profiling of PGCs
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Total DNA from FACS-sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and DNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 26 µL final volume using the Savant SpeedVac Concentrator (Thermo) and following the manufacturer’s instructions. Genomic DNA was digested by 20 units of MspI enzyme (NEB) in NEB buffer 2 at 37°C for 3 hrs, and digested DNA was purified using AMPure XP beads (Beckman-Coulter). Libraries were made following the NEBNext Ultra DNA Library Prep protocol with methylated adaptors and the following modifications: following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma); and PCR enrichment was carried out for 18 cycles using the KAPA Uracil+ DNA polymerase master mix (KAPA Biosystems) and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Pooled libraries were sequenced on the Illumina HiSeq 2500 instrument, using the 'dark sequencing' protocol, as previously described32 (link).
7
DNA Extraction and Bisulfite Sequencing
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The genomic DNA of FACS isolated YFP-positive cells was extracted by using DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer’s instructions. Then genomic DNA was digested by MspI enzyme (NEB) and size selected using the EpiNext™DNA Size Selection Kit (EpigenTek) to enrich the DNA fragment between 100-600 bp. The selected DNA fragments were used to prepared into sequencing library with EpiNext™High-Sensitivity Bisulfite-Seq Kit (EpigenTek) according to manufacturer’s instructions.
8
miR-196a2 Genotyping by PCR-RFLP
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Total DNA was extracted from EDTA treated blood samples using Zymo Quick-gDNA™ MiniPrep DNA Purification Kit (Zymo Research, CA, USA).
After ethanol precipitation, the DNA was purified and dissolved in double distilled water and frozen at -20 °C until use. The miR-196a2 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). The PCR primers (Thermo scientific) were as follows: forward 5′-CCCCTTCCCTTCTCCTCCAGATA-3′ and reverse 5′-CGAAAACCGACTGATGTAACTCCG-3′. PCR cycling conditions were 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 63 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 10 min. For restriction fragment length polymorphism, the PCR products were digested with 5 units MsPI enzyme (New England Biolabs, USA) at 37 °C and visualized by electrophoresis on 2% agarose under ultraviolet (UV) illumination. The allele types were determined as follows: a single 149 bp fragment for the TT genotype, 2 fragments of 24 and 125 bp for the CC genotype, and 3 fragments of 24, 125, and 149 bp for the TC genotype.
After ethanol precipitation, the DNA was purified and dissolved in double distilled water and frozen at -20 °C until use. The miR-196a2 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). The PCR primers (Thermo scientific) were as follows: forward 5′-CCCCTTCCCTTCTCCTCCAGATA-3′ and reverse 5′-CGAAAACCGACTGATGTAACTCCG-3′. PCR cycling conditions were 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 63 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 10 min. For restriction fragment length polymorphism, the PCR products were digested with 5 units MsPI enzyme (New England Biolabs, USA) at 37 °C and visualized by electrophoresis on 2% agarose under ultraviolet (UV) illumination. The allele types were determined as follows: a single 149 bp fragment for the TT genotype, 2 fragments of 24 and 125 bp for the CC genotype, and 3 fragments of 24, 125, and 149 bp for the TC genotype.
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