All flash-frozen tissue was freeze-dried for one day, then stored at −80°C until DNA extraction. DNA was extracted with the HP Plant DNA mini kit (Omega Bio-Tek, protocol version May 2013) from ≥ 5 mg of lyophilized leaf tissue after grinding with a mixer-mill (Retsch MM 400) in 2 mL Eppendorf tubes with tungsten beads supplied (60 s at 30 Hz). We added 10 µl beta-mercaptoethanol at step 2, and equilibrated the columns at step 8 of the standard protocol. The extracted DNA was eluted in 2 × 100 µl EB buffer (Qiagen), cleaned, and concentrated with the Clean and Concentrator-5 kit (Zymo Research, protocol v1.2.1) using 15,000 g for all centrifugation steps, and finally eluted in 2 × 30 µl EB buffer (Qiagen) at 60°C.
Eb buffer
Manufactured by Qiagen
Sourced in Germany, United States
EB buffer is a laboratory solution used in various DNA and RNA extraction and purification protocols. It serves as an elution buffer to facilitate the release of the target nucleic acid from the extraction matrix. The composition and formulation of EB buffer are designed to maintain the integrity and stability of the extracted nucleic acids.
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Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (12 mentions)
Dneasy blood and tissue kit, by Qiagen (11 mentions)
Ampure xp bead, by Beckman Coulter (11 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Hiseq 2000, by Illumina (7 mentions)
Nextseq 500, by Illumina (7 mentions)
Hiseq 4000, by Illumina (7 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
Nextseq 550, by Illumina (5 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (5 mentions)
Hiseq 2500, by Illumina (5 mentions)
Minelute pcr purification kit, by Qiagen (5 mentions)
Pe buffer, by Qiagen (5 mentions)
Kapa library quantification kit, by Roche (5 mentions)
Dneasy blood tissue kit, by Qiagen (5 mentions)
Proteinase k, by New England Biolabs (5 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Agencourt ampure xp bead, by Beckman Coulter (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (4 mentions)
220 protocols using eb buffer
1
Freeze-Dried Leaf DNA Extraction
2
16S rRNA Gene Amplification and Sequencing
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The 16S rRNA gene was amplified using primers 515F-Y (5′-GTGYCAGCMGCCGCGGTAA) and 926R (5′-CCGYCAATTYMTTTRAGTTT), which target the V4–V5 region (Pyke et al. 2023 (link)). The PCR reaction protocol was as follows: 12.5 µl of KAPA Hi-Fi polymerase, 0.15 µl each of forward and reverse primers, 0.625 µl of bovine serum albumin (BSA), 9.5 µl PCR-grade H2O, and ~5 ng of DNA template. Thermocycling conditions used for 16S rRNA amplification were as follows: 95°C for 3 min, followed by 25 cycles of 95°C for 2 s, 58°C for 20 s, 72°C for 15 s, followed by a final extension at 72°C for 5 min. Indexing PCR reactions were identical to 16S rRNA amplification, reducing to eight cycles and removing the BSA additive. All samples (208) were purified using NuceloMag magnetic bead purification (Macherey-Nagel, Düren Germany) following the manufacturer’s protocol, and final purified amplicons were eluted in 50 µl of EB Buffer (Qiagen). Amplicons from indexing PCR reactions were purified using the same method as the initial amplification and eluted in 50 µl of EB Buffer (Qiagen). All samples were quantified using PicoGreen (ThermoFisher) using the manufacturer’s protocol. Samples were then normalized using a Hamilton Liquid Handling Robot (Hamilton Company, NV, USA) and pooled into amplicon libraries for loading onto an Illumina MiSeq Next-Generation Sequencer.
3
Automated PCR-Free DNA Library Prep
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For each sample, 500 ng of DNA were diluted in a total volume of 60 μL of EB buffer (Qiagen, Hilden, Germany). DNA was then transferred to a 96-well plate and sheared for 1 h using the Covaris Focused-ultrasonicator. DNA was purified using Ampure XP in 20% polyethylene glycol (PEG) beads to obtain fragments sized from 200 to 500 basepairs (Beckman-Coulter, Brea, USA). Library preparation was performed on the Bravo Automated Liquid Handling Platform (Agilent, Santa Clara, USA) using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, USA). Following end-repair and A-tailing, adapters were ligated overnight at room temperature. PCR-free library preparation allowed for the conservation of methylated cytosines for subsequent methylated DNA immunoprecipitation. Finally, DNA was resuspended in 35 μL of EB buffer (Qiagen, Hilden, Germany). DNA was quality controlled using Qubit Fluorometric Quantitation and the DNA 1000 Bioanalyzer 2100 kit (Agilent, Santa Clara, USA) to verify DNA concentration and fragment size (250–550 bp).
4
Viral Genome Conformation Capture
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V3C-seq assays were performed with Hind III as the primary restriction enzyme to digest cross-linked MVM infected A9 fibroblast chromatin. The Hind III-digested DNA was intramolecular-ligated using the 3C procedure, before resuspending in Buffer EB (100 µ l, Qiagen). 3C-DNA was secondary-digested with Nla III (100U, overnight at 37°C), before being heat inactivated and circularized with 100U of T4 DNA Ligase at room temperature overnight in 6 ml of ligation reaction. The V3C samples were precipitated by phenol:chloroform extraction, precipitated in isopropanol, resuspended in Qiagen Buffer EB (100 µ l), and. Inverse PCR was performed on the circularized DNA using primers within the Hind III - Nla III fragments on the MVM genome using inverse PCR primers described in Supplementary file 1 . Inverse PCR products were diluted 1:100 in TE buffer and used as templates for nested inverse PCRs (described in Supplementary file 1 ), yielding V3C-seq DNA libraries. Sequencing libraries were prepared using the NEB Ultra Kit, and twelve samples were pooled per run for 75 base-pair single end sequencing using an Illumina Next Seq 500 sequencer.
5
Viral DNA Extraction and Quantification
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Patient plasma samples were split into two fractions and treated either with Buffer RDD (Qiagen) or DNAse I (Qiagen). DNA detected from the DNAse I-treated fraction represents only virion-encapsidated DNA, while mock-treated DNA (by Buffer RDD) comprises the total DNA in the patient’s plasma (cell-free host DNA, virion-encapsidated DNA and free-floating viral DNA). An equal volume of cold phenol-saturated Tris-EDTA (10 mM Tris, pH 8.0 with 1 mM EDTA) was added to each tube followed by vigorous shaking. Samples were then centrifuged at 13,000 relative centrifugal force (rcf) at 4°C for 15 mins. An additional 100 μL of Buffer EB (Qiagen) was gently added to facilitate aspiration. The aqueous phase of DNA was extracted and stored at -20°C until use. Standard ethanol precipitation using glycogen as carrier was performed to concentrate the DNA. Buffer EB (Qiagen) was used to resuspend the purified DNA. Virus DNA quantification was performed in accordance to published qPCR protocol using 1 μL template DNA (31 (link)). Standard curve was generated using DNA from the Namalwa EBV-positive Burkitt’s lymphoma cell line (32 (link)).
6
Protocol for Quantifying gDNA for HIV Assays
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Measure the concentration of the gDNA with a NanoDrop or other spectrophotometer.
If the gDNA is >200 ng/μL and <300 ng/ul after the EtOH precipitation, dilute with Qiagen Buffer EB to a maximum of 200 ng/μL for the HIV assays. If the gDNA is >300 ng/ul, repeat BglI digestion.
Prepare at least 20 μL of 1:100 diluted gDNA in Qiagen Buffer EB or molecular grade water. This will be used for the two replicate wells of the reference assay.
Store the gDNA at 4°C until ready to proceed with ddPCR.
7
Targeted Manipulation of Fos mRNA and ecRNA
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To manipulate Fos mRNA or ecRNA levels, we designed four 20 bp ASOs that targeted distinct transcripts from the Fos gene locus (Fig. 6a ; Supplementary Data 3 ). ASOs targeting exon 3 of Fos mRNA or Fos 3′ ecRNA were synthesized with two chemical modifications: an all phosphorothioate backbone and five 2′ O-methyl RNA bases on each end of the oligonucleotide (Integrated DNA Technologies). These modifications have been shown to enhance ASO stability and improve affinity for the target RNA molecule51 (link), and have successfully been employed for targeted manipulations in animal models of degenerative and developmental disorders52 (link)53 (link). Primary neuronal cultures were treated with vehicle (15 μl buffer EB, Qiagen) or ASO (15 μl in buffer EB, for a final concentration of 1.5 μM) and incubated for 72 h. Following ASO treatment, RNA was extracted (Qiagen RNeasy kit) and Fos mRNA and ecRNA levels were determined using RT-qPCR with custom primers (Supplementary Data 3 ). Gene expression was determined by normalizing to Gapdh (2−ΔΔCt).
8
Single-cell DNA extraction and PCR
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Single cells were selected from raw samples, washed three or four times in sterile mineral water, placed individually in 0.2 ml PCR tube with 25 μl of EB buffer (Qiagen, Valencia, CA, USA) and stored at − 20 °C. Cells were not starved prior to selection. DNA was extracted from each of five cells using a modified Chelex method [45 (link)]. PCR was done as follows: in 0.2 ml PCR tubes, we used 12.5 μL GoTaq® Green Master Mix, 2× (Madison, WI, USA), 1.25 μl each of universal eukaryotic forward primer EUK-A and reverse primer EUK-B [46 (link)], both in a final concentration of 0.4 μM, and 10.5 μl of the Chelex extraction for a total volume of 25 μl. PCR was performed in the iCycler™ Thermal Cycler and DNA sequencing in both directions was done at GENEWIZ (South Plainfield, NJ, USA) as previously described [47 (link)]. Contigs were assembled by Seqman (DNAStar).
9
DNA Quantification and Purification Protocol
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The DNA concentration of the samples was measured using the Qubit dsDNA BR Assay kit (Invitrogen). DNA (1–2 µl) was added to Qubit dsDNA BR working solution such that the total volume was 200 µl. The mixture was vortexed for 2–3 s, ensuring that no bubbles formed, and was then incubated for 2–5 min at room temperature prior to the measurement of DNA concentration with a Qubit 2.0 fluorometer (Invitrogen).
DNA purification of amplicons was carried out using the Agencourt AMPure XP system (Beckman Coulter). Room temperature AMPure XP beads were mixed with DNA samples in low DNA-binding microcentrifuge tubes (Eppendorf), after which the mixtures were incubated for 5 min at room temperature. The tubes were then placed next to the magnets on the magnet rack to sit until all the beads were pulled towards the magnet. The supernatant was then removed, after which the beads were washed twice with 1 ml of 80 % ethanol while the tubes were still on the rack. After the supernatant was removed, the beads were allowed to air dry for 5 min on the magnet to allow evaporation of excess ethanol. The tubes were removed from the magnets, and DNA was eluted with EB buffer (Qiagen) The DNA samples were quantified after purification.
DNA purification of amplicons was carried out using the Agencourt AMPure XP system (Beckman Coulter). Room temperature AMPure XP beads were mixed with DNA samples in low DNA-binding microcentrifuge tubes (Eppendorf), after which the mixtures were incubated for 5 min at room temperature. The tubes were then placed next to the magnets on the magnet rack to sit until all the beads were pulled towards the magnet. The supernatant was then removed, after which the beads were washed twice with 1 ml of 80 % ethanol while the tubes were still on the rack. After the supernatant was removed, the beads were allowed to air dry for 5 min on the magnet to allow evaporation of excess ethanol. The tubes were removed from the magnets, and DNA was eluted with EB buffer (Qiagen) The DNA samples were quantified after purification.
10
Ancient DNA Analysis of Mungyeong Mummy
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This study was declared by the Institutional Review Board (IRB) of Seoul National University Hospital as an exempt (IRB no. 2013–004). It was conducted in accordance with the Vermillion Accord on Human Remains, World Archaeological Congress (South Dakota, 1989).
The brain tissue of the Mungyeong mummy was subjected to aDNA analysis. The sample (0.5–1 g) was incubated in 4 mL of lysis buffer (EDTA 50 mM, pH 8.0; 1 mg/mL of proteinase K; SDS 1%; 0.1M DTT) at 56°C for 24 hr. Total DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and then treated with chloroform/isoamyl alcohol (24:1). DNA isolation and purification was performed using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The purified DNA was eluted in 50 μl of EB buffer (QIAGEN) [26 (link),27 (link)].
The brain tissue of the Mungyeong mummy was subjected to aDNA analysis. The sample (0.5–1 g) was incubated in 4 mL of lysis buffer (EDTA 50 mM, pH 8.0; 1 mg/mL of proteinase K; SDS 1%; 0.1M DTT) at 56°C for 24 hr. Total DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and then treated with chloroform/isoamyl alcohol (24:1). DNA isolation and purification was performed using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The purified DNA was eluted in 50 μl of EB buffer (QIAGEN) [26 (link),27 (link)].
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