For WGBS library construction, 300ng genomic DNA was isolated from FACS-sorted HSCs (as indicated above, and performed in duplicates) and fragmented using a Covaris sonication system (Covaris S2). DNA libraries were constructed using the Illumina TruSeq DNA sample preparation kit. After ligation, libraries were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen, Valencia, CA). Ligation efficiency was tested by PCR using TrueSeq primers and Pfu TurboCx hotstart DNA polymerase (Stratagene). After determining the optimized PCR cycle number for each sample, a large scale PCR reaction (100 ul) was performed as described previously40 (link). PCR products were sequenced with Illumina HiSeq sequencing systems. Paired- end bisulfite-treated sequencing reads were aligned to the mouse genome mm9. The adapters and the low-quality reads were trimmed using BSMAP with default. Sequencing depth of CpGs at least 5 reads of each sample were extract to calculate the methylation ratio calling [0,1] by the software MOABS61 (link). A two-state first-order hidden Markov model was used to detect the undermethylated regions and larger undermethylated canyons (≥ 3.5 Kb) as described previously40 (link).
Sonication system
Manufactured by Covaris
Sourced in United States
The Sonication system by Covaris is a device designed for sample preparation through the use of focused acoustic energy. The core function of this equipment is to disrupt cellular structures and macromolecules, such as DNA, RNA, and proteins, through the application of controlled sonication.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq sequencing system, by Illumina (3 mentions)
Truseq dna sample preparation kit, by Illumina (3 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Agilent bioanalyzer analysis, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Dna clean concentrator 5 column, by Zymo Research (1 mentions)
Pfuturbo cx hotstart dna polymerase, by Agilent Technologies (1 mentions)
Unmethylated lambda dna, by Promega (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Epitech bisulfite kit, by Qiagen (1 mentions)
Pacbio sequel instrument, by Pacific Biosciences (1 mentions)
Magbead kit, by Pacific Biosciences (1 mentions)
Megaruptor 2, by Diagenode (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
7 protocols using sonication system
1
WGBS Library Construction and Methylation Analysis for HSCs
2
Sperm DNA Methylation Profiling
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Sperm DNA of nine men (three men randomly selected from each of the RL, RM, and RH groups) aged 20–40 years were submitted for MeDIP and hMeDIP sequencing. These men did not have a history of smoking or alcohol intake, their blood Cd concentration was < 1 µg/dL, and BPA, DEHP, MEHP, and DBP concentrations were < 1 µg/µmolCr. The sperm DNA was extracted from samples using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. Agarose gel electrophoresis was used to detect the integrity of genome, Nano drop (Thermo Scientific) was used to detect DNA purity (OD 260/280 ratio), and the DNA concentration was measured using Qubit 2.0 fluorometer (Life Technologies). The extracted DNA was fragmented using the Covaris sonication system, and sequencing libraries were prepared from the 5-μg fragments of genomic DNA. End repair, A base addition, and adaptor ligation steps were performed using the Illumina’s Single-End DNA sample preparation kit. Adaptor-ligated DNA was immunoprecipitated by anti-5mC and anti-5hmC by using a commercial antibody. The immunoprecipitated DNA was purified and then applied for 50-bp single-end sequencing on the Illumina Hiseq2500 platform.
3
Methyl-DNA Immunoprecipitation Sequencing
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All sample preparation and MeDIP-sequencing was performed by the BGI-Shenzhen, Shenzhen, China. Extracted DNA was fragmented using a Covaris sonication system and sequencing libraries were prepared from 5 μg fragmented genomic DNA. End repair, base addition and adaptor ligation steps were performed using Illumina’s Single-End DNA Sample Prep kit. Adaptor-ligated DNA was immunoprecipitated by anti-5mC using a commercial antibody (Diagenode), and MeDIP products were validated by quantitative PCR. MeDIP DNA was purified with ZYMO DNA Clean & Concentrator-5 columns, and amplified using adaptor-mediated PCR. DNA fragments between 220 and 320 bp in size were gel-excised, and amplification quality and quantity were evaluated by Agilent BioAnalyzer analysis. The libraries were subjected to highly parallel 50 bp single-end sequencing on the Illumina HiSeq platform.
4
Genomic DNA Methylation Profiling
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Genomic DNA was extracted from a pool of 50 individuals from each of the four life stages: 3 days embryo, late larva, 3 days pupa and 3 days adult, using the DNeasy Blood & Tissue Kit (Qiagen, CA). Then, 5 µg of genomic DNA from each sample spiked with 25 ng of unmethylated Lambda DNA (Promega, Madison, WI, USA) was fragmented to a size of 100–300 bp with a Covaris sonication system (Covaris, MA), followed by blunt end, 3′-end addition of dA. Cytosine-methylated adaptors were ligated to the fragmented DNA using T4 DNA ligase according to the manufacturer’s instructions. Bisulphite conversion of the sample DNA was performed using a ZYMO EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer’s instructions. The converted DNA was then amplified through nine cycles of PCR with PfuTurbo Cx Hotstart DNA Polymerase (Agilent Technologies, CA).
5
Whole-Genome Bisulfite Sequencing Protocol
6
WGBS Library Construction and Methylation Analysis for HSCs
7
Illumina and PacBio Sequencing for Genomic Assembly
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Genomic DNA was fragmented using a Covaris sonication system. Short-insert paired-end libraries (350 bp) were constructed according to Illumina’s protocol with end repair, poly-A tail base addition, sequencing-adaptor ligation, amplification, and purification. Paired-end sequencing was performed with an Illumina HiSeq X Ten platform (Illumina, USA). The Illumina raw reads were evaluated using FASTQC v0.11.6 [45 ] and filtered with fastp v0.20 [46 (link)] using the default parameters to produce the Illumina clean reads for subsequent analysis.
Simultaneously, the extracted genomic DNA was sheared using Megaruptor2 (Diagenode, Ougrée, Belgium), and then used to construct SMRT bell libraries via the ligation of universal hairpin adaptors onto double-stranded DNA fragments in accordance with the 20-kb preparation protocol (Pacific Biosciences, USA). The MagBead kit (Pacific Biosciences, USA) was used to remove adaptor dimers. The failed ligation products were removed using exonucleases. The sequencing primer was annealed to each SMRT bell template for subsequent sequencing with a PacBio Sequel instrument using Sequel Sequencing Kit 1.2.1 (Pacific Biosciences, USA). The PacBio polymerase reads were filtered using the RS_Subreads protocol with minimum length > 300 bp to produce the PacBio subreads for genome assembly.
Simultaneously, the extracted genomic DNA was sheared using Megaruptor2 (Diagenode, Ougrée, Belgium), and then used to construct SMRT bell libraries via the ligation of universal hairpin adaptors onto double-stranded DNA fragments in accordance with the 20-kb preparation protocol (Pacific Biosciences, USA). The MagBead kit (Pacific Biosciences, USA) was used to remove adaptor dimers. The failed ligation products were removed using exonucleases. The sequencing primer was annealed to each SMRT bell template for subsequent sequencing with a PacBio Sequel instrument using Sequel Sequencing Kit 1.2.1 (Pacific Biosciences, USA). The PacBio polymerase reads were filtered using the RS_Subreads protocol with minimum length > 300 bp to produce the PacBio subreads for genome assembly.
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