Amplified DNA was quantified by gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and randomly sheared by ultrasound sonication (Covaris M220) to produce fragments of ≤ 800 bp. The sticky ends were repaired, and adapters were added using T4 DNA polymerase (M4211, Promega, USA), Klenow DNA polymerase (KP810250, Epicentre), and T4 polynucleotide kinase (EK0031, Thermo Fisher Scientific, USA). Fragments of 300–800 bp were collected after electrophoresis. After amplification, libraries were pooled and subjected to 150-bp, 250-bp, or 300-bp paired-end sequencing on the NovaSeq 6000, HiSeq X Ten, and MiSeq platforms (Illumina, USA). Considering that the RT-WGA libraries were likely to have higher virus diversity than the WGA and WTA libraries [100 ], they were sequenced with higher depth and thus produced better assembly results (Table S1 ).
Klenow dna polymerase
Manufactured by Illumina
Sourced in United States
Klenow DNA Polymerase is a DNA-dependent DNA polymerase enzyme derived from the large fragment of E. coli DNA Polymerase I. It catalyzes the polymerization of nucleotides into DNA strands in a template-dependent manner.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by Thermo Fisher Scientific (3 mentions)
T4 dna polymerase, by Promega (3 mentions)
T4 dna polymerase, by Illumina (3 mentions)
Hiseq x ten, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Truseq dna preparation protocol, by Illumina (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Repli g cell wga wta kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Qiaquick spin column, by Qiagen (1 mentions)
Minelute column, by Qiagen (1 mentions)
Genomic dna sample prep kit, by Illumina (1 mentions)
Hiscansq, by Illumina (1 mentions)
Rnaseh, by Illumina (1 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
7 protocols using klenow dna polymerase
1
Optimized DNA Fragmentation and Sequencing Protocol
2
Viral Metagenomic Sequencing from Fecal Samples
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Fecal samples were processed using a previously published method [22 (link),23 (link)]. Total RNA was extracted from enriched virus-like particles using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The extracted RNA of all samples was pooled for library construction, followed by amplification using the REPLI-g Cell WGA & WTA Kit (150052; Qiagen, Hilden, Germany). Amplified DNA was randomly fragmented by ultrasound sonication (Covaris M220, Woburn, MA, USA) to produce 800 bp fragments, then sticky ends were repaired and adapters were added using T4 DNA polymerase (M4211, Promega, Madison, WI, USA), Klenow DNA Polymerase (KP810250, Epicentre, Woburn, MA, USA), and T4 polynucleotide kinase (EK0031, Thermo Scientific, Fermentas, GlenBurnie, MD, USA). Each viral sequencing library was prepared following the Illumina TruSeq DNA Preparation Protocol and was sequenced on the HiSeq4000 platform (Illumina, San Diego, CA, USA), with 150 bp paired ends. The library preparation and sequencing process was performed by BGI Tech (Shenzhen, China).
3
Whole-Genome Sequencing Library Preparation
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The amplified DNA was quantified using gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Ultrasonic random shearing (Covaris M220) was performed to generate fragments with lengths ≤800 bp. Fragment ends were repaired using T4 DNA Polymerase (M4211, Promega, Madison, Wisconsin), Klenow DNA Polymerase (KP810250, Epicentre, Madison, Wisconsin), and T4 Polynucleotide Kinase (EK0031, Thermo Fisher Scientific, Waltham, MA). Fragments in the range of 300–800 bp were collected after electrophoresis. After amplification, the libraries were pooled, and paired-end sequencing of 150 bp, 250 bp, or 300 bp was performed on the Novaseq 6000, HiSeq X ten, and Miseq platforms (Illumina, San Diego, California) (Patch et al., 2018 (link); Ravi et al., 2018 (link); Modi et al., 2021 (link)).
4
Illumina Whole-Genome Chromatin IP Sequencing
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The purified DNA products were modified for Illumina Whole-Genome Chromatin IP sequencing using an Illumina Genomic DNA Sample Prep kit as follows: size-selected DNAs were end-repaired by T4 DNA polymerase and phosphorylated by T4 DNA polymerase and T4 polynucleotide kinase. The products were incubated with Klenow DNA Polymerase (Illumina) to generate 3′ adenine overhangs and then ligated to Illumina adapters, which contain 5′ thymine overhangs. The adapter-ligated products were purified on QIAquick spin columns (Qiagen), PCR-amplified with Phusion DNA Polymerase (Finnzymes) for 30 cycles using an Illumina genomic DNA primer set. The PCR products were purified on QIAquick and MinElute columns (Qiagen).The quality of the DNA was assessed and quantified using an Agilent DNA 1000 Series II assay and NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and the DNA was diluted to 10 nM. Cluster generation and sequencing were performed using a Standard Cluster Generation kit and a Cycle Solexa Sequencing kit on the Illumina Cluster Station and Illumina HiSeq2000 sequencer following the manufacturer's instructions. Sequencing was carried out by the Research & Cooperation Division, BGI-Shenzhen.
5
cDNA Library Construction and Sequencing
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For cDNA library construction, 2 μg of total RNA were treated with 1U of DNaseI amplification grade (Invitrogen) and purified according to the TruSeq Stranded mRNA Sample Prep LS Protocol of Illumina (http://grcf.jhmi.edu/hts/protocols/mRNA-Seq_SamplePrep_1004898_D.pdf ), using magnetic microspheres for messenger RNA separation. The purified mRNA was fragmented in Illumina buffer. Superscript III (Invitrogen) and oligo(dT) were used for reverse transcription of the first cDNA strand. The second strand was synthesized using the enzymes RNase H and DNA Polymerase I (Illumina). Molecule ends were treated with T4 DNA Polymerase and Klenow DNA Polymerase (Illumina), making them blunt. The 3’ end of the synthetized cDNA was phosphorylated with T4 PNK (Illumina) and adenylated with Klenow exo (Illumina). Adaptors were bound to cDNA ends, and the samples were purified and selected by size of 200 bp ± 25 bp after fractioning in agarose gel electrophoresis (QIAquick Gel Extraction Kit, QIAGEN). Purified cDNA was quantified by RT-qPCR using adaptor-specific oligonucleotides (Illumina).
Sequencing was performed using HiScanSQ (Illumina),—according to the manufacturer’s recommendations, and using the paired-end reads protocol. Each sample was sequenced until it reached around 34 million reads/library.
Sequencing was performed using HiScanSQ (Illumina),—according to the manufacturer’s recommendations, and using the paired-end reads protocol. Each sample was sequenced until it reached around 34 million reads/library.
6
Illumina DNA Library Preparation
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Large DNA fragments were sheared into small fragments (≤800 bp) using a high throughput sonication instrument (Covaris or BioRuptor). The sticky end of the small DNA fragments was converted into a blunt end using T4 DNA Polymerase, Klenow DNA Polymerase and T4 polynuleotide kinase (Illumina, Inc., San Diego, CA, USA), followed by adaptors ligating to the ends. Subsequently, the blunt-ended DNA fragments were subjected to electrophoretic separation (2% agarose gel in TAE buffer; 120V; 60 min) to recover the target DNA products, followed by polymerase chain reaction amplification according to the manufacturer's instructions. Briefly, the PCR reaction mix included DNA (1 µg), Phusion DNA polymerase (Finnzymes; Thermo Fisher Scientific, Inc., Waltham, MA, USA), PCR primer 1.1 (1 µl; Illumina, Inc.), PCR primer 2.1 (1 µl; Illumina, Inc.) and deionized water (22 µl). Amplify protocols were as follows: 98°C for 30 sec, 10 cycles of 98°C for 10 sec, 65°C for 30 sec, and 72°C for 30 sec, with a final extension at 72°C for 5 min. When the DNA library was ready, DNA clusters were formed and sequenced on an Illumina HiSeq 2000 instrument (Illumina, Inc.).
7
ChIP-seq Library Preparation Protocol
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