Wizard dna clean up system

We used the method of Liu et al.^{24 (link)} for DNA extraction and sodium bisulfite treatment, without any modifications. We isolated genomic DNA using a TIANamp Genomic DNA kit (Tiangen, Beijing, China) and checked for purity of DNA using NanoDrop 1,000 Spectrophotometer (Thermo Fisher Scientific, Shanghai, China). 2 µg of DNA was denatured (in 3 M NaOH for half hour at 42 °C), followed by incubation with freshly prepared 2.5 M sodium bisulfite and 1 M hydroquinone in a total volume of 520 µl at 55 °C for 16 h. Purification of DNA was done using Wizard DNA Clean-Up System (Promega, Madison, WI, USA). The reaction was terminated by the addition of 5.5 µl NaOH (3 M) at room temperature for 15 min. Sample was precipitated by the addition of 33 µl ammonium acetate (10 M, pH 7.0), 270 µl ethanol and 4.0 µl glycogen (10 µg/µl), at − 20 °C for 12 h. The modified DNA was resuspended in 30 µl elution buffer and stored at − 20 °C.

HCC cells and tissue samples were digested with proteinase K. Then genomic DNA was isolated by phenol-chloroform extraction. The extracted DNA was diluted in TE buffer and stored at −20°C. 2 μg of genomic DNA was diluted in 50 μl of water. The bisulfite treatment was carried out for 16 h at 50°C. DNA samples were then purified with the Wizard DNA Clean-Up System (Promega, WI, USA), desulfonated with NaOH, and then precipitated with ethanol and resuspended in 20 μl of water.

A mixture of 4 μg genomic DNA, 10 μg salmon sperm DNA and 0.3 M NaOH was collected. Water was used to top up the final volume to 20 μl. The mixture was incubated at 50 °C for 20 min to denature the DNA. Next, the mixture was transferred into 500 μl of solution containing 3 M sodium bisulfite (Sigma, Saint Louis, MO) and 10 mM hydroquinone (Sigma, Saint Louis, MO) and incubated at 70 °C for 4 h. DNA was then purified using the Wizard DNA Clean-Up System (Promega Corp., Madison, WI). After purification, DNA was followed by ethanol precipitation, dry and dissovled in distilled water.

We used a modified cetyltrimethylammonium bromide (CTAB) method to extract high-quality DNA (Doyle and Doyle, 1987 ), which was then purified using the Wizard^® DNA cleanup system (Promega, Madison, WI, USA). DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), and DNA integrity was evaluated by electrophoresis on a 1% (w/v) agarose gel. A DNA library was prepared using the NEB Next Ultra DNA Library Prep Kit for Illumina (San Diego, CA, USA). Libraries for paired-end 150-bp sequencing were analyzed on an Illumina NovaSeq 6000 platform (Novogene Co., Ltd., Tianjin, China) to generate approximately 10 GB of data for each sample. Raw reads were filtered using SOAPnuke to remove sequencing adaptors and low-quality bases (Chen et al., 2018 (link)). The filtered reads were assembled using GetOrganelle (Jin et al., 2020 (link)) with a range of 21, 45, 65, 85, and 105 k-mers for plastomes and 35, 85, and 115 k-mers for nrDNA. Subsequently, ITS and ETS sequences were uploaded to the NCBI GenBank database (accession numbers are listed in Table 2).

At least 10⁷ MCF7 and LTED cells were crosslinked using 1% formaldehyde in medium for 5 min at room temperature with rotation. Cells were lysed using NP-40 lysis buffer for 15 min at 4 °C. Nuclei were washed and re-suspended in restriction buffer and then digested with 1 U/μL HindIII (TaKaRa, 1060AH) overnight at 37 °C while shaking. After inactivating the restriction enzymes by heating at 65 °C for 20 min in the presence of 1.6% sodium dodecyl sulfate (SDS), proximity ligation was done in 7 mL total volume using 350 U T4DNA ligase (TaKaRa, 2011A) for 4 h at 16 °C and then kept for 30 min at room temperature. After de-crosslinking and RNase treatment, the DNA was purified by phenol–chloroform extraction and ethanol precipitation. The 3C template was cleaned using the wizard DNA cleanup system (Promega, A7280). The ligated products (3C template) were assessed using qPCR with the ABI Prism 7300 and Step One Plus system (Applied Biosystems) using TaqMan Fast advanced mastermix (ABI). Primers in this analysis are listed in Supplementary Table 6. For the standard qPCR curve, we used purified BAC DNAs that were digested and ligated. The BAC DNAs containing the target genomic loci were ESR1p-BAC for reference and another locus position for a–d (see “BAC clones” section for details).

Genomic DNA from the Salmonella phages was extracted using the Wizard DNA Clean-Up system (A7280; Promega, Madison, WI, USA) following the modified Promega Wizard method, as described by the Center for Phage Technology, Texas A&M University, USA [32 (link)]. Extracted DNA was purified by ethanol precipitation [33 ] and whole-genome sequencing (WGS) was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) with 300 bp paired-end libraries and 30× coverage. Raw sequence reads were assembled using the A5 pipeline [34 (link)] and genome annotation was completed using the Bacterial and Viral Bioinformatics Resource Center (formerly PATRIC) [35 (link),36 (link)]. Annotations were manually curated and the coding sequences (CDSs) were used to interrogate the NCBI database using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins (accessed on 6 January 2024)) [37 (link)]. An HHpred search of the Pfams database was used to identify conserved protein motifs [38 (link)]. To assign a protein to a gene sequence, at least 90% identity was sought in BLASTP searches for protein motifs [39 (link)]. Based on the presence or absence of a gene encoding integrase, phages were putatively classified as temperate or virulent, respectively [26 (link)].

To determine the genome type of halovirus VOLN27B, the genome of VOLN27B was isolated from the virus stock according to the method described by Summer [23 (link)] using Wizard DNA Clean-Up System (Promega, USA). Genomic DNA (1 μg) was digested with DNase I (5 U) (TaKaRa, Japan), Exonuclease III (20 U) (TaKaRa, Japan), and Mung Bean Nuclease (4 U) (TaKaRa, Japan) at 37°C for 1 h, and the resulting product was checked by DNA electrophoresis. An equal amount of undigested plasmid pBR322 (NEB, USA) was used as a circular dsDNA standard, and the EcoRI (TaKaRa, Japan) cleaved pBR322 was used as a linear dsDNA standard. These untreated and EcoRI leaved pBR322 plasmids were also used as controls in treatments nucleases DNase I, Exonuclease III, and Mung Bean Nuclease.