In situ Hi-C was performed according to a published protocol11 (link). Briefly, two million cells were cross-linked with 1% formaldehyde for 10 mins at room temperature. Reaction was then quenched with 0.2M Glycine. Cell pellets were washed with cold PBS and lysed with lysis buffer to get nuclei pellets. Nuclei were permeabilized with 0.5% SDS. DNA was in situ digested with 100 units of DpnII (NEB, R0543) overnight. The ends of restriction fragments were filled with biotinylated nucleotides (Invitrogen, 19524016) and in situ ligated with T4 DNA ligase (NEB, M0202). After reversal of crosslinks, ligated DNA was ethanol precipitated and sheared to about 400bp by sonication (Covaris). Sonicated products were pulled down with streptavidin beads. Library construction was then performed on beads. After adapter ligation, beads were suspended in 20ul TE buffer and subjected to bisulfite conversion with EZ DNA Methylation-Gold™ Kit (Zymo, D5005). Unmethylated lambda DNA (Promega, D1501) was sonicated and ligated with the same adapter for Methyl-HiC sample and then was spiked in at 0.5% before bisulfite conversion. Purified bisulfite converted DNA was amplified with HiFi Hotstart Uracil+Ready Mix (KAPA, KK2802).
Hifi hotstart uracil readymix
Manufactured by Roche
Sourced in United States
HiFi HotStart Uracil+ ReadyMix is a high-fidelity, ready-to-use PCR master mix designed for amplification of DNA or cDNA templates. It contains a thermostable DNA polymerase, dNTPs, and buffer components optimized for efficient and accurate DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Epitect bisulfite kit, by Qiagen (3 mentions)
Biotinylated nucleotides, by Thermo Fisher Scientific (2 mentions)
Dpnii, by New England Biolabs (2 mentions)
Unmethylated lambda dna, by Promega (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (2 mentions)
Pyromark q24, by Qiagen (2 mentions)
Library amplification primer mix, by Roche (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
2100 bioanalyzer, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Tapestation 4000, by Agilent Technologies (1 mentions)
9 protocols using hifi hotstart uracil readymix
1
In Situ Hi-C Methylome Profiling Protocol
2
In Situ Hi-C Methylome Profiling Protocol
3
Bisulfite Sequencing for DNA Methylation
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Genomic DNA underwent bisulfite conversion using EpiTect Bisulfite Kit (Qiagen) following the manufacturer’s instructions. Regions of interest were amplified with specific PCR primers (Supplementary Table 7 ) using HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems). For clonal Sanger sequencing, amplified regions were cloned into pCR4-TOPO vector (Invitrogen) and more than 15 bacterial clones were used for Sanger sequencing. For pyrosequencing, amplified regions were subjected to pyrosequencing reaction in PyroMark Q24 (Qiagen) with specific sequencing primers (Supplementary Table 7 ) following the manufacturer’s instructions. The data were analyzed with the PyroMark Q24 software (Qiagen).
4
KAPA Double-Stranded DNA Library Prep
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The KAPA double-stranded DNA library preparations were performed manually at the AFMES-AFDIL using the Roche KAPA Hyper Prep Kit. Unless otherwise noted, library preparation was carried out according to the manufacturer protocol. Again, 5 µL of extract was used for library preparation, and a positive control and negative control were included with each sample set. The positive control was fragmented DNA from a K562 cell line with 1 ng library input. End repair and A-tailing took place in a single step with incubation at 20 °C for 30 min, then 65 °C for 30 min (lid 85 °C), followed by adapter ligation using 15 µM KAPA dual indexed adapters for all libraries at 20 °C for 15 min. Post-ligation clean-up used an increased 2× AMPure XP ratio in order to retain smaller fragments. The full library volume was amplified using KAPA HiFi HotStart Uracil+ ReadyMix and KAPA Library Amplification Primer mix, according to the manufacturer’s recommendations with the following parameters: 95 °C for 3 min, then 35 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 1 min, followed by 72 °C for 7 min. The same post-amplification AMPure XP purification was utilized as with the SRSLY libraries. Libraries were generated from a total of 15 samples, one RB, two positive controls and two negative controls.
5
Bisulfite Sequencing Library Prep
6
Enzymatic Methyl-seq Library Preparation
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Libraries were prepared using the NEBNext Enzymatic Methyl-seq (NEB, E7120S) kit, following the manufacturer's instructions. 50, 5, 2, or 0.5 ng of 5mC adaptor-ligated NIH3T3 DNA was used as input. HiFi HotStart Uracil + Ready Mix (KAPA Biosystems, KK2801) was used to amplify the libraries following conversion before purification over SPRI beads (0.8× left-sided) and elution in nuclease-free water to yield final libraries. Libraries were then quantified by Qubit HS (Invitrogen, Q32851) and quality checked on an Agilent Bioanalyzer 2100 before sequencing on an Illumina MiSeq instrument to confirm conversion efficiencies. Details of each input's elution size and library amplification cycles are listed in Supplementary Table S1C .
7
Mouse Liver DNA Methylation Profiling
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Genomic DNA was isolated from snap-frozen mouse liver tissues and digested with 20 units of MspI (New England Biolabs, Ipswich, MA, USA). After purification, the blunt-end DNA fragments were added with dA, followed by methylated-adapter (Illumina, San Diego, CA, USA) ligation. The libraries were size selected for fractions in 40–120 bp and 120–220 bp range on a 2% agarose gel, and then subjected to bisulfite conversion using EpiTect Bisulfite Kit (Qiagen, Valencia, CA, USA). The final libraries were amplified using HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems, Wilmington, MA, USA) and Illumina Multiplexing PCR Primers. Quality and quantity of RRBS libraries were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) and reverse transcriptase–PCR, respectively.
8
Bisulfite Sequencing for DNA Methylation
9
Whole Genome Enrichment and Sequencing
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Between 100 -500ng of library amplified DNA was subjected to in-solution target enrichment using whole human genome myBaits® WGE (Arbor Biosciences) as detailed in Wasef et al. (Wasef et al., 2018) and Wright et al. (Wasef et al., 2018; Wright et al., 2018) . Target capture enrichment steps were performed according to manufacturer's instructions with the following modifications: the hybridisation step was performed for 36 -42 hrs at 57°C. The beads, and the bead binding buffers were heated to 57°C for 30 min before being used. Further cleaning steps were also performed at the same hybridisation temperature. Post-capture libraries were amplified on beads using HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems) for between 14 and 17 cycles, and then visualised using the 5000 High-Sensitivity DNA tapes on the TapeStation 4000 (Agilent Technologies).
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