Following digestion with TaqI (10 U, New England Biolabs®), the mixture was cooled on ice, and then 10 U PstI HF (New England Biolabs®) was added and the reaction incubated for an additional 1 h at 37 °C. Subsequently, the pre-annealed specific adaptors (Syntezza Bioscience®) (Table S2) were ligated to the genomic DNA fragments using T4 DNA ligase (1 U, MBI Fermentas®). Ligation was done at 22 °C for 3 h, and the resulting RL products used as templates in AFLP PCR without heat inactivation of endonucleases.
Psti hf
Manufactured by New England Biolabs
Sourced in United States
PstI-HF is a type II restriction endonuclease that recognizes and cleaves the DNA sequence 5'-CTGCAG-3'. It is a high-fidelity variant of the traditional PstI enzyme, providing enhanced specificity and reducing star activity. PstI-HF is commonly used in molecular biology applications such as DNA cloning, mapping, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (6 mentions)
Bamhi hf, by New England Biolabs (5 mentions)
Ecori hf, by New England Biolabs (4 mentions)
Nextseq 500, by Illumina (2 mentions)
Pippin prep, by Sage Science (2 mentions)
Hiseq x ten pe150 platform, by Illumina (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Dsdna br assay, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nucleospin tissue, by Macherey-Nagel (1 mentions)
Dna sequencing, by Takara Bio (1 mentions)
Stellar competent cells, by Takara Bio (1 mentions)
Saci hf, by New England Biolabs (1 mentions)
Taq dna polymerase, by Tiangen Biotech (1 mentions)
Tianprep mini plasmid kit, by Tiangen Biotech (1 mentions)
Escherichia coli top10 cells, by Tiangen Biotech (1 mentions)
31 protocols using psti hf
1
Genomic DNA Fragmentation and Adapter Ligation
2
ddRAD-seq Library Preparation for Illumina
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Genomic DNA was extracted from adipose fin clips using a column extraction kit (NucleoSpin Tissue, Macherey Nagel, Düren, Germany) following the manufacturer’s recommended protocols. DNA quality was assessed by visualization after gel electrophoresis on a 2% Agarose gel and quantified using a Qubit 2.0 Fluorometer with the dsDNA BR Assay (Life Technologies, Carlsbad, CA, USA). Each sample was normalized to a total amount of 1 µg of DNA with a minimum concentration of 25 ng/µL. DNA was used to construct a single ddRAD-library containing 44 individuals, following a previously described protocol in [67 (link)] with a modified set of barcoded P1 and P2 adapters for paired-end sequencing on Illumina platforms. In brief, each sample was digested using two restriction enzymes, a rare-cutting enzyme (PstI-HF, 20 units) and a frequent-cutting enzyme (MspI, 20 units) (New England Biolabs, Ipswich, MA, USA). Samples were individually barcoded using unique combinations of barcoded P1 and P2 adapters. After multiplexing of the barcoded samples, a size selection step was performed using the Pippin Prep (Sage Science, Beverly, MA, USA) and fragments between 145–295 bp were selected. The amplified library was sequenced on half a lane (200 million reads) of an Illumina NextSeq 500 (75 bp paired-end reads) at Glasgow Polyomics (University of Glasgow).
3
Cloning and Validation of ncRNA Plasmids
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Individual human tRNA sequences were obtained from the Genomic tRNA Database (GtRNAdb), and human miRNA sequences were extracted from miRBase. Plasmids encoding target ncRNAs (Table S1 ) were cloned as previously reported 28 (link) involving two strategies. Most inserts were obtained through PCR amplification using htRNA-specific primers (Table S2 ) (IDT, San Diego, CA) and corresponding btRNAMet-based ncRNA expression plasmids24 as templates. A few other target inserts were directly amplified through PCR reactions with longer primers containing 16-nt complementary sequences (Table S2 ). The amplicons were thus cloned into pBSTNAV 29 (link) linearized by endonucleases EcoRI-HF® and PstI-HF® (New England Biolabs, Ipswitch, MA) as previously described 28 (link). All clones were propagated in Stellar™ Competent Cells (Takara Bio, Mountain View, CA) and confirmed by DNA sequencing (Genscript, Piscataway, NJ).
4
Cloning and Expression of EsMBD Genes
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The cloning and restriction primers were designed using Prime premier 5.0 (Supplementary Table S7) [39 (link)]. The EsMBD sequences were cloned using cloning primers and enzyme digestion primers, using E. senticosus cDNA as a template and Taq DNA polymerase (TIANGEN, Beijing). The amplification conditions were as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30s, annealing at 55 °C for 30s, extension at 72 °C for 5 min, and an additional extension at 72 °C for 5 min. A lethal fast cloning kit (TIANGEN, Beijing) was used to connect the cloned sequences with the pLB vectors and was introduced into competent Escherichia coli Top10 cells (TIANGEN, Beijing, China). Sequencing was performed using a SinoGenoMax (Beijing, China). The recombinant pLB plasmids were extracted using a TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China). The pHG vectors and recombinant pLB plasmids were digested overnight at 37 °C with BamHI-HF, PstI-HF, and SacI-HF (NEW ENGLAND BIOLABS, Beijing, China) restriction enzymes. We used T4 DNA ligase (NEW ENGLAND BIOLABS, Beijing, China) to connect EsMBD genes with pHG vectors overnight at 16 °C to construct recombinant EsMBD-GFP expression vectors. The total reaction system was 20.0 µL with 1.0 µL T4 DNA ligase, 1.0 µL Nuclease-Free Water, 2.0 µL T4 DNA ligase Buffer, 11.0 µL Insert DNA (20 ng / µL), 5.0 µL pHG vector.
5
Constructing Genomic Libraries using Super-GBS
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we adopted modified GBS methods (SuperGBS, following Qi et al. [43 (link)]) to construct libraries. Briefly, extracted DNA from each individual was digested with both PstI-HF and MspI (New England Biolabs, NEB) for 2 h at 37 °C and then 2 h incubation at 75 °C. The barcoded adapters and common adapter were respectively ligated on the PstI cut site and the MspI cut site of all samples by T4 DNA ligase (NEB), ligation was running at 22 °C for 2 h. Fragments smaller than 300 bp were removed using recovery system of improved magnetic bead. The recovered fragment was amplified by PCR using high-fidelity enzymes, before libraries were sequenced, the concentration of PCR product was tested by Qubit2.0 and it should be greater than 5 ng/ul. Final libraries were sequenced using Illumina Hiseq Xten, PE150 Platform.
6
Bacterial Genetic Interactions via BACTH
7
Genome-wide SNP Discovery via GBS in Cucumis
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To obtain genome-wide SNPs, GBS analyses [24 (link)] were conducted for 91 di- and 102 tetraploid individuals, with one of the latter included twice as a replicate. For the library preparation, 200 ng of genomic DNA was used and cut with the two restriction enzymes PstI-HF (NEB) and MspI (NEB). Library preparation, individual barcoding, and single-end sequencing on the Illumina NovaSeq were performed following Wendler et al. [28 (link)].
Barcoded reads from the 194 samples were de-multiplexed using theCasava pipeline 1.8 (Illumina). Adapter trimming of GBS sequence reads was performed with Cutadapt [29 (link)] within ipyrad v.0.9.58 [30 (link)] and reads shorter than 60 bp after adapter removal were discarded. GBS reads were clustered using the ipyrad 0.7.5 [30 (link)] pipeline with a clustering threshold of 0.85. We tested diverse ipyrad settings but at the end the default settings of parameter files generated with ipyrad were optimal for the other parameters. We generated one output that included the outgroup C. malyi, which was used for phylogenetic analyses, and a second output without C. malyi, which was used for principal component analysis (PCA) and population assignment analyses.
Barcoded reads from the 194 samples were de-multiplexed using the
8
Assessing Nucleosome Fidelity in Libraries
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MN fidelity in the final, assembled MN library was determined using a modified version of the restriction enzyme accessibility assay. This assay, described in the ACF remodeling assays section below, enables determination of rates of MN sliding based on liberation of a PstI restriction site embedded in the nucleosome 601 positioning sequence. The restriction enzyme digestion employed in this assay can also be used to quantify the fraction of disassembled nucleosomes for each individual MN in the library. Aliquots of the DNA-barcoded MN library were treated with or without 2 U/μL PstI-HF (New England Biolabs) at 37 °C for 64 min. Barcoded DNA was amplified and prepared for and subjected to next-generation sequencing as described above. MN barcode read counts were first normalized among samples to the ‘uncuttable’ (PstI site absent) non-nucleosomal DNA barcode read counts. The ‘cuttable’ (PstI site present) non-nucleosomal DNA barcode served as a positive control for PstI digestion. These data confirmed that MNs in the library were well assembled (98 ± 5%) (Extended Data Fig. 2d ).
9
Cloning and Expression of GFP
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The CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain intermediate constructs by direct PCR and restriction/ligation cloning of required genetic elements. The pRSET-EmGFP plasmid (Thermo Fisher Scientific) was used as an expression vector containing the GFP gene. High-fidelity restriction endonucleases BamHI-HF, BglII-HF, EcoRI-HF, NcoI-HF, and PstI-HF; T4-DNA-ligase; T4-polynucleotide-kinase; and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs (Ipswich, MA, USA). The oligonucleotides were synthesised by the solid-phase method and purified by preparative polyacrylamide gel electrophoresis (PAGE) by Syntol LLC (Russia). Q5® High-Fidelity DNA Polymerase (New England Biolabs) was used for all polymerase chain reaction (PCR). Ultrafree-DA Centrifugal Filter Units (Merck, Kenilworth, NJ, USA) were used for DNA extraction from agarose gel. The ZymoPURE™ Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) was used for plasmid DNA purification. The authenticity of the plasmids was confirmed by Sanger sequencing performed by Eurogen CJSC (Russia). E. coli strain NiCo21(DE3) (#C2529H, New England Biolabs, Ipswich, MA, USA) was used for cloning and expression experiments according to the manufacturer’s protocol.
10
Rapid Sequencing Library Preparation
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Three RAD libraries with twelve individuals each were prepared following a modified RAD sequencing protocol [1 (link)], using PstI-HF (New England BioLabs) restriction enzyme to digest 300 ng of genomic DNA per sample. Digested DNA was ligated to P1 barcoded adapters using twelve different barcodes for each library. Adapter-ligated fragments were pooled and sheared targeting a 500 bp average fragment size using a sonicator. To remove adapter dimers, libraries were purified with Agencourt AMPure XP (Beckman Coulter) magnetic beads after P2 adapter ligation with a volume DNA/beads ratio of 1:0.8. After end-repair using a commercial kit (New England BioLab), libraries were amplified by Polymerase Chain Reaction (PCR) performing an initial denaturation step at 98°C for 30 s, followed by 18 cycles of one denaturation step at 98°C for 10 s, annealing at 65°C for 30 s, extension at 72°C for 30 s and a final 5 min extension step. PCR-enriched libraries were purified with AMPure XP beads and the DNA concentration of each library was quantified in a Qubit 2.0 (Invitrogen). Libraries, in a proportional representation, were paired end sequenced in three lanes of an Illumina HiSeq 2000 at Genepool (Ashworth Laboratories).
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