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SDCpro-GFP expression and <t>DNA</t> demethylation caused by R175Q mutation in MTHFD1 . ( a ) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/ mthfd1-2 F1 and #162/ mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2 . Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. ( b ) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding 28 . ( c ) <t>PCR-based</t> genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/ mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. ( d ) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. ( e ) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. ( f ) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. ( n =3). Different letters above bars indicate significant differences between pairwise comparisons by Student's t -test ( P
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1) Product Images from "MTHFD1 controls DNA methylation in Arabidopsis"

Article Title: MTHFD1 controls DNA methylation in Arabidopsis

Journal: Nature Communications

doi: 10.1038/ncomms11640

SDCpro-GFP expression and DNA demethylation caused by R175Q mutation in MTHFD1 . ( a ) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/ mthfd1-2 F1 and #162/ mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2 . Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. ( b ) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding 28 . ( c ) PCR-based genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/ mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. ( d ) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. ( e ) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. ( f ) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. ( n =3). Different letters above bars indicate significant differences between pairwise comparisons by Student's t -test ( P
Figure Legend Snippet: SDCpro-GFP expression and DNA demethylation caused by R175Q mutation in MTHFD1 . ( a ) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/ mthfd1-2 F1 and #162/ mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2 . Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. ( b ) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding 28 . ( c ) PCR-based genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/ mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. ( d ) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. ( e ) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. ( f ) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. ( n =3). Different letters above bars indicate significant differences between pairwise comparisons by Student's t -test ( P

Techniques Used: Expressing, Mutagenesis, Fluorescence, Binding Assay, Polymerase Chain Reaction, Methylation, Microelectrode Array

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Amplification:

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Synthesized:

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Construct:

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Incubation:

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Modification:

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Hybridization:

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Flow Cytometry:

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Ligation:

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Generated:

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Sequencing:

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DNA Extraction:

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RNA Sequencing Assay:

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Magnetic Beads:

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Isolation:

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Size-exclusion Chromatography:

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Purification:

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Article Snippet: The library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA). .. The DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail (Illumina Inc., San Diego, CA, USA) in a 10-cycle polymerase chain reaction (PCR).

Article Title: Transcriptome Analysis of Portunus trituberculatus in Response to Salinity Stress Provides Insights into the Molecular Basis of Osmoregulation
Article Snippet: In order to select cDNA fragments of preferentially 200 bp in length the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR reaction.

Article Title: RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes
Article Snippet: To select cDNA fragments of preferentially 200 bp in length the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR reaction.

Article Title: Elucidation of the molecular responses to waterlogging in Sesbania cannabina roots by transcriptome profiling
Article Snippet: .. To select cDNA fragments of the preferred 200 bp in length, the library fragments were purified using an AMPure XP system (Beckman Coulter, Beverly, CA, USA). cDNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction. .. The products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay on the Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA).

Article Title: Identification of MicroRNAs and Their Target Genes Associated with Ovarian Development in Black Tiger Shrimp (Penaeus monodon) Using High-Throughput Sequencing
Article Snippet: In brief, RNA bands at ~20–30 bp were separated and purified by 6% Tris/Borate/EDTA (TBE) polyacrylamide gel electrophoresis (PAGE) and subsequently bound to 3′and 5′end adapters in two separate steps, followed by PAGE purification. .. After first strand cDNA synthesis using random oligonucleotides and SuperScript™ II, DNA fragments ligated with adapters on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction.

Article Title: Whole Transcriptomic Analysis Provides Insights into Molecular Mechanisms for Toxin Biosynthesis in a Toxic Dinoflagellate Alexandrium catenella (ACHK-T)
Article Snippet: .. In order to select cDNA fragments of preferentially 200 bp length, adapter ligated fragments were purified and enriched using an Illumina PCR Primer Cocktail in a 10 cycle PCR reaction to create the final libraries. .. The quality and quantity of the cDNA libraries were measured using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: .. Libraries were then amplified for sequencing using 2× Robust Master Mix (KAPA), 10× PCR Primer Cocktail (Illumina) and half the volume of resuspended streptavidin beads, for 12 cycles, purified using 0.8× volumes of AMPure XP beads, then sequenced. .. Sequencing was carried out using Illumina HiSeq 2000 and NextSeq 500 instruments to generate paired-end 80 bp or paired-end 101 bp reads.

Article Title: Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
Article Snippet: The solution was incubated for 5 min at 55°C, followed by purification with DNA Clean & Concentrator-5 (Zymo Research) according to the Nextera user manual. .. Elution was performed with 25 μL of resuspension buffer (RSB) from the Nextera kit, and 20 μL was used for the final enrichment PCR along with 15 μL of Nextera PCR master mix (NPM), 5 μL of Index 1 primers (N7xx), 5 μL of Index 2 primers (N5xx), and 5 μL of PCR primer cocktail (PPC; composed of FC-121-1012 and FC-121-1011 primers; see Illumina website).

Article Title: Hypoxia causes transgenerational impairments in reproduction of fish
Article Snippet: Index codes were ligated to identify the individual samples. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Illumina, San Diego, USA) before fragmentation using divalent cations under elevated temperature in Illumina fragmentation buffer. .. DNA fragments successfully ligated with adaptor molecules on both ends were enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR reaction.

Polymerase Chain Reaction:

Article Title: Identification of the key genes and long non-coding RNAs in ankylosing spondylitis using RNA sequencing
Article Snippet: .. Subsequently, the Uracil-N-Glycosylase enzyme was added into the purified ligation product and incubated at 37°C for 10 min. A total of 15 rounds of polymerase chain reaction (PCR) amplification were conducted with PCR Primer Cocktail (Illumina, Inc.) and PCR Master Mix (Illumina, Inc.) to enrich the cDNA fragments. .. The PCR products were then purified with AMPure XP beads (Qiagen, Inc.).

Article Title: Staphylococcus aureus induces COX-2-dependent proliferation and malignant transformation in oral keratinocytes
Article Snippet: .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail via a 15-cycle PCR. .. The products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay on the Bioanalyzer 2100 system (Agilent).

Article Title: Comparative transcriptomics identifies patterns of selection in roses
Article Snippet: .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. .. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).

Article Title: Evolution of DNA Methylation Patterns in the Brassicaceae is Driven by Differences in Genome Organization
Article Snippet: .. Reaction conditions are all follows: 32.9 µl of water, 5 µl of 10× Pfu Cx Buffer, 5 µl of 2 mM dNTP, 1.6 µl of Illumina PCR Primer Cocktail, 0.5 µl of Cx Polymerase (2.5 U/µl), 5 µl of bisulfite-treated DNA eluate. .. Three PCR reactions were pooled for each bisulfite-treated sample.

Article Title: Integrated Transcriptomic, Proteomic, and Metabolomics Analysis Reveals Peel Ripening of Harvested Banana under Natural Condition
Article Snippet: .. The DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail (Illumina Inc., San Diego, CA, USA) in a 10-cycle polymerase chain reaction (PCR). .. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000 platform (Illumina Inc).

Article Title: Transcriptome Analysis of Portunus trituberculatus in Response to Salinity Stress Provides Insights into the Molecular Basis of Osmoregulation
Article Snippet: .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR reaction. .. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.

Article Title: RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes
Article Snippet: .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR reaction. .. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.

Article Title: Elucidation of the molecular responses to waterlogging in Sesbania cannabina roots by transcriptome profiling
Article Snippet: .. To select cDNA fragments of the preferred 200 bp in length, the library fragments were purified using an AMPure XP system (Beckman Coulter, Beverly, CA, USA). cDNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction. .. The products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay on the Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA).

Article Title: Identification of MicroRNAs and Their Target Genes Associated with Ovarian Development in Black Tiger Shrimp (Penaeus monodon) Using High-Throughput Sequencing
Article Snippet: .. After first strand cDNA synthesis using random oligonucleotides and SuperScript™ II, DNA fragments ligated with adapters on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction. .. Products of 145–160 bp (with adaptors on both sides) were separated by PAGE and quantified with the Agilent 2100 bioanalyzer.

Article Title: Whole Transcriptomic Analysis Provides Insights into Molecular Mechanisms for Toxin Biosynthesis in a Toxic Dinoflagellate Alexandrium catenella (ACHK-T)
Article Snippet: .. In order to select cDNA fragments of preferentially 200 bp length, adapter ligated fragments were purified and enriched using an Illumina PCR Primer Cocktail in a 10 cycle PCR reaction to create the final libraries. .. The quality and quantity of the cDNA libraries were measured using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: .. Libraries were then amplified for sequencing using 2× Robust Master Mix (KAPA), 10× PCR Primer Cocktail (Illumina) and half the volume of resuspended streptavidin beads, for 12 cycles, purified using 0.8× volumes of AMPure XP beads, then sequenced. .. Sequencing was carried out using Illumina HiSeq 2000 and NextSeq 500 instruments to generate paired-end 80 bp or paired-end 101 bp reads.

Article Title: Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
Article Snippet: .. Elution was performed with 25 μL of resuspension buffer (RSB) from the Nextera kit, and 20 μL was used for the final enrichment PCR along with 15 μL of Nextera PCR master mix (NPM), 5 μL of Index 1 primers (N7xx), 5 μL of Index 2 primers (N5xx), and 5 μL of PCR primer cocktail (PPC; composed of FC-121-1012 and FC-121-1011 primers; see Illumina website). .. The PCR program was as follows: 3 min at 72°C, 30 sec at 98°C, and then five cycles of 10 sec at 98°C, 30 sec at 63°C, and 3 min at 72°C.

Article Title: MTHFD1 controls DNA methylation in Arabidopsis
Article Snippet: .. Libraries were amplified using PCR primer cocktail (Illumina) and Pfu Turbo Cx hotstart DNA polymerase (Agilent). .. Sequencing was performed on a HiSeq 2000 platform at 50 bp length.

Article Title: Hypoxia causes transgenerational impairments in reproduction of fish
Article Snippet: .. DNA fragments successfully ligated with adaptor molecules on both ends were enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. .. Products were purified using the AMPure XP system and quantified using the Agilent Bioanalyzer 2100 system.

Polyacrylamide Gel Electrophoresis:

Article Title: Identification of MicroRNAs and Their Target Genes Associated with Ovarian Development in Black Tiger Shrimp (Penaeus monodon) Using High-Throughput Sequencing
Article Snippet: In brief, RNA bands at ~20–30 bp were separated and purified by 6% Tris/Borate/EDTA (TBE) polyacrylamide gel electrophoresis (PAGE) and subsequently bound to 3′and 5′end adapters in two separate steps, followed by PAGE purification. .. After first strand cDNA synthesis using random oligonucleotides and SuperScript™ II, DNA fragments ligated with adapters on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction.

cDNA Library Assay:

Article Title: Identification of the key genes and long non-coding RNAs in ankylosing spondylitis using RNA sequencing
Article Snippet: Subsequently, the cDNA library for RNA sequencing was contrasted using the TruSeq RNA Sample Prep kit (Illumina, Inc.). .. Subsequently, the Uracil-N-Glycosylase enzyme was added into the purified ligation product and incubated at 37°C for 10 min. A total of 15 rounds of polymerase chain reaction (PCR) amplification were conducted with PCR Primer Cocktail (Illumina, Inc.) and PCR Master Mix (Illumina, Inc.) to enrich the cDNA fragments.

Article Title: Transcriptome Analysis of Portunus trituberculatus in Response to Salinity Stress Provides Insights into the Molecular Basis of Osmoregulation
Article Snippet: Paragraph title: RNA Isolation, cDNA Library Construction and Illumina Deep Sequencing ... DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 10 cycle PCR reaction.

Article Title: Elucidation of the molecular responses to waterlogging in Sesbania cannabina roots by transcriptome profiling
Article Snippet: Paragraph title: RNA isolation, cDNA library construction, and sequencing ... To select cDNA fragments of the preferred 200 bp in length, the library fragments were purified using an AMPure XP system (Beckman Coulter, Beverly, CA, USA). cDNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction.

Sample Prep:

Article Title: Identification of the key genes and long non-coding RNAs in ankylosing spondylitis using RNA sequencing
Article Snippet: Subsequently, the cDNA library for RNA sequencing was contrasted using the TruSeq RNA Sample Prep kit (Illumina, Inc.). .. Subsequently, the Uracil-N-Glycosylase enzyme was added into the purified ligation product and incubated at 37°C for 10 min. A total of 15 rounds of polymerase chain reaction (PCR) amplification were conducted with PCR Primer Cocktail (Illumina, Inc.) and PCR Master Mix (Illumina, Inc.) to enrich the cDNA fragments.

Article Title: Comparative transcriptomics identifies patterns of selection in roses
Article Snippet: Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction.

Article Title: Identification of MicroRNAs and Their Target Genes Associated with Ovarian Development in Black Tiger Shrimp (Penaeus monodon) Using High-Throughput Sequencing
Article Snippet: Small RNA library construction and sequencing According to the protocol of the Illumina TruSeq Small RNA Sample Preparation Kit (Illumina), 3 μg pooled RNA was used for sRNA library construction. .. After first strand cDNA synthesis using random oligonucleotides and SuperScript™ II, DNA fragments ligated with adapters on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 12-cycle PCR reaction.

Article Title: Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
Article Snippet: Paragraph title: Tagmentation reaction and final PCR amplification using Nextera and Nextera XT DNA sample preparation kits ... Elution was performed with 25 μL of resuspension buffer (RSB) from the Nextera kit, and 20 μL was used for the final enrichment PCR along with 15 μL of Nextera PCR master mix (NPM), 5 μL of Index 1 primers (N7xx), 5 μL of Index 2 primers (N5xx), and 5 μL of PCR primer cocktail (PPC; composed of FC-121-1012 and FC-121-1011 primers; see Illumina website).

Article Title: Hypoxia causes transgenerational impairments in reproduction of fish
Article Snippet: Briefly, the cDNA libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA) following the manufacturer's recommendations. .. DNA fragments successfully ligated with adaptor molecules on both ends were enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR reaction.

Spectrophotometry:

Article Title: Staphylococcus aureus induces COX-2-dependent proliferation and malignant transformation in oral keratinocytes
Article Snippet: The quality and concentration of the RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific), and the integrity was confirmed by a Bioanalyzer 2100 system (Agilent). mRNA was purified from the high-quality total RNA using poly-T oligo-attached magnetic beads. .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail via a 15-cycle PCR.

Concentration Assay:

Article Title: Staphylococcus aureus induces COX-2-dependent proliferation and malignant transformation in oral keratinocytes
Article Snippet: The quality and concentration of the RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific), and the integrity was confirmed by a Bioanalyzer 2100 system (Agilent). mRNA was purified from the high-quality total RNA using poly-T oligo-attached magnetic beads. .. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail via a 15-cycle PCR.

High Throughput Screening Assay:

Article Title: Identification of the key genes and long non-coding RNAs in ankylosing spondylitis using RNA sequencing
Article Snippet: Paragraph title: Library preparation and high-throughput sequencing ... Subsequently, the Uracil-N-Glycosylase enzyme was added into the purified ligation product and incubated at 37°C for 10 min. A total of 15 rounds of polymerase chain reaction (PCR) amplification were conducted with PCR Primer Cocktail (Illumina, Inc.) and PCR Master Mix (Illumina, Inc.) to enrich the cDNA fragments.

Lysis:

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Immobilized DNA was precipitated via DynaMag, washed once with 600 μL 0.5× Bind and Wash buffer mixed with 0.5× TE lysis buffer (25 mM Tris–HCl, 0.5 mM EDTA, 0.5 % SDS), once with 600 μL 1× Bind and Wash buffer, once with 600 μL 1× NEBuffer #2, once with 600 μL Buffer EB (10 mM Tris–HCl pH 8.5), and resuspended in 20 μL Buffer EB. .. Libraries were then amplified for sequencing using 2× Robust Master Mix (KAPA), 10× PCR Primer Cocktail (Illumina) and half the volume of resuspended streptavidin beads, for 12 cycles, purified using 0.8× volumes of AMPure XP beads, then sequenced.

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    Illumina Inc nextera dna library preparation kit
    Nextera Dna Library Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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