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End repair mix

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End-repair mix is a laboratory reagent used to prepare DNA fragments for subsequent processing, such as ligation or sequencing. It is designed to convert the ends of DNA molecules to a state that is compatible with downstream applications. The core function of the end-repair mix is to enzymatically modify the ends of DNA fragments to create blunt ends or specific overhang structures as required by the application.

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Lab products found in correlation

T4 dna ligase, by Qiagen (11 mentions) Nextseq 500, by Illumina (10 mentions) Spriselect beads, by Beckman Coulter (8 mentions) Klenow exo, by Qiagen (6 mentions) Dynabeads oligo dt 25, by Thermo Fisher Scientific (5 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions) Trizol reagent, by Thermo Fisher Scientific (4 mentions) Trizol, by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Turbofect, by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) Klenow exonuclease minus, by Qiagen (3 mentions) Q5 dna polymerase, by New England Biolabs (3 mentions) Minelute column, by Qiagen (3 mentions) Ampure xp, by Beckman Coulter (3 mentions) Platinum taq dna polymerase, by Thermo Fisher Scientific (3 mentions) Dna oligonucleotides, by Merck Group (3 mentions) Bioruptor 0.5 ml microtubes for dna shearing, by Diagenode (3 mentions) T4 dna ligase for guide seq, by Qiagen (3 mentions)

21 protocols using end repair mix

1

Prostate Cancer TERT Methylation MeDIP-Seq

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For in-depth analysis of the TERT gene methylation status MeDIP-Seq was used [30 (link)]. Library preparation 2.5 μg of genomic DNA from 51 prostate cancers or 53 normal prostate tissues were fragmented to 100 to 200 bp using the Covaris S2 system and end repaired with End Repair mix (Enzymatics) followed by a purification step (Qiagen DNA Purification Kit) and ligation of barcoded SOLiD sequencing adapters as previously described [30 (link)].
Castelo-Branco P., Leão R., Lipman T., Campbell B., Lee D., Price A., Zhang C., Heidari A., Stephens D., Boerno S., Coelho H., Gomes A., Domingos C., Apolonio J.D., Schäfer G., Bristow R.G., Schweiger M.R., Hamilton R., Zlotta A., Figueiredo A., Klocker H., Sültmann H, & Tabori U. (2016). A cancer specific hypermethylation signature of the TERT promoter predicts biochemical relapse in prostate cancer: a retrospective cohort study. Oncotarget, 7(36), 57726-57736.
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2

Strand-Specific RNA-Seq Library Preparation

Cited 3 times
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Total RNA was extracted from U2932 cells using RNeasy Mini Kit (QIAGEN, #74104) and polyA+ transcripts isolated with oligo (dT)25-conjugated magnetic Dynabeads (Thermo Fisher). Strand specific RNA-seq libraries were prepared following a published protocol62 (link). Briefly, RNA was chemically fragmented in first strand buffer, converted to cDNA using SuperScript® III reverse transcriptase (Invitrogen), end-repaired, A-tailed and ligated to custom-designed universal adapters using an end-repair mix, klenow fragment, and T4 DNA ligase (all from Enzymatics). After ligation, adapters were removed by SPRI purification using SPRIselect beads (Beckman coulter) and amplified with Q5 Hot Start DNA polymerase (New England Biolabs) while introducing custom dual indexes. Three biological replicates were sequenced on a NextSeq 500 (Illumina) at a depth of at least 2×107 reads each. Reads were mapped and analyzed with a custom bioinformatic pipeline based on STAR63 (link), SAMTOOLS64 (link), and the R packages DEGseq65 (link) and DEseq266 (link). We used human genome version GRCh38 and gene annotations from the ENSEMBL release 83. GO analyses were performed using version 6.8 of the DAVID web server67 (link), 68 (link).
Choi J., Lee K., Ingvarsdottir K., Bonasio R., Saraf A., Florens L., Washburn M.P., Tadros S., Green M.R, & Busino L. (2018). Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2. Nature cell biology, 20(5), 586-596.
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3

Strand-Specific RNA-Seq Library Preparation

Cited 3 times
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Total RNA was extracted from U2932 cells using RNeasy Mini Kit (QIAGEN, #74104) and polyA+ transcripts isolated with oligo (dT)25-conjugated magnetic Dynabeads (Thermo Fisher). Strand specific RNA-seq libraries were prepared following a published protocol62 (link). Briefly, RNA was chemically fragmented in first strand buffer, converted to cDNA using SuperScript® III reverse transcriptase (Invitrogen), end-repaired, A-tailed and ligated to custom-designed universal adapters using an end-repair mix, klenow fragment, and T4 DNA ligase (all from Enzymatics). After ligation, adapters were removed by SPRI purification using SPRIselect beads (Beckman coulter) and amplified with Q5 Hot Start DNA polymerase (New England Biolabs) while introducing custom dual indexes. Three biological replicates were sequenced on a NextSeq 500 (Illumina) at a depth of at least 2×107 reads each. Reads were mapped and analyzed with a custom bioinformatic pipeline based on STAR63 (link), SAMTOOLS64 (link), and the R packages DEGseq65 (link) and DEseq266 (link). We used human genome version GRCh38 and gene annotations from the ENSEMBL release 83. GO analyses were performed using version 6.8 of the DAVID web server67 (link), 68 (link).
Choi J., Lee K., Ingvarsdottir K., Bonasio R., Saraf A., Florens L., Washburn M.P., Tadros S., Green M.R, & Busino L. (2018). Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2. Nature cell biology, 20(5), 586-596.
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4

RNA Isolation and Sequencing Protocol

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Cultured cells were rinsed once with PBS and lysed via direct addition of TRIzol reagent (Thermo Fisher Scientific). Total cellular RNA was purified using the Direct-zol RNA Miniprep kit (Zymo Research) or via PCIA. For RT-qPCR, 25 ng of RNA were assayed per 10 μL reaction using the RNA-to-Ct single-step kit (Thermo Fisher) and Gapdh as a normalization control. RT-qPCR primers are listed in the STAR key resources table. For library preparation, polyA+ RNA was isolated from 1.5 μg (ESC and NPC experiments) or 400 ng (CP experiments) total RNA using Dynabeads Oligo (dT)25 beads (Thermo Fisher) and constructed into strand-specific libraries using the dUTP method. UTP-marked cDNA was end-repaired using end-repair mix (Enzymatics), tailed with deoxyadenine using Klenow exo- (Enzymatics), and ligated to custom dual indexed adapters with T4 DNA ligase (Enzymatics). Libraries were size-selected with SPRIselect beads (Beckman Coulter) and quantified by qPCR after amplification. Paired-end sequencing was performed on a NextSeq 500 (Illumina).
Petracovici A, & Bonasio R. (2021). Distinct PRC2 subunits regulate maintenance and establishment of Polycomb repression during differentiation. Molecular cell, 81(12), 2625-2639.e5.
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5

Chromatin Profiling and Transcriptomics in mESCs

Cited 2 times
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CUT&RUN was performed as previously described (30 (link),31 (link)) using EZH2, H3K27me3 and IgG antibodies on 5 million cells per sample. DNA obtained from Drosophila melanogaster S2 cells was sonicated to 200bp and 1ng was added to each CUT&RUN sample as a heterologous spike-in control. ChIP was performed as previously described (32 (link)) using 9 μg of ATRX antibody (21 (link)) and 6 million cells per sample. For RNA-sequencing, RNA samples were extracted from Day 0 mESCs and Day 6 NPCs using Trizol reagent (Invitrogen) and subjected to DNase digestion with Turbo DNase (Ambion AM2238), then rRNA-depleted using FastSelect -rRNA HMR (Qiagen) and converted to cDNA using Ultra II Directional RNA Library Prep Kit (NEB E7760). DNA and cDNA samples were end-repaired using End-Repair Mix (Enzymatics), A-tailed using Klenow exonuclease minus (Enzymatics), purified with MinElute columns (Qiagen), and ligated to Illumina adapters (NEB #E7600) with T4 DNA ligase (Enzymatics). Size selection for fragments >150 bp was performed with AMpure XP (Beckman Coulter). Libraries were PCR amplified with barcoded adapters for Illumina sequencing (NEB #E7600) using Q5 DNA polymerase (NEB #M0491) and purified with MinElute. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38 × 2 paired-end cycles.
Bieluszewska A., Wulfridge P., Doherty J., Ren W, & Sarma K. (2022). ATRX histone binding and helicase activities have distinct roles in neuronal differentiation. Nucleic Acids Research, 50(16), 9162-9174.
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6

Salivary Gland RNA-Seq of Drosophila Mtor KD

Cited 1 time
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Ten salivary glands per sample were collected from wandering 3rd instar larvae of wild-type (Nub-Gal4 x w118) and Mtor KD flies in 1 mL of TRIzol (Ambion). Total RNA was purified as mention above and the quality of RNA was examined by running on agarose-formaldehyde gels. For library preparation, polyA+ RNA was isolated from 500 ng of total RNA using Oligo(dT)25 Dynabeads (Thermo Fisher) and constructed into strand-specific libraries using the dUTP method (Parkhomchuk et al., 2009 (link)). UTP-marked cDNA was end-repaired using end-repair mix (Enzymatics, MA), tailed with deoxyadenine using Klenow exo- (Enzymatics), and ligated to custom dualindexed adapters with T4 DNA ligase (Enzymatics). Libraries were size-selected with SPRIselect beads (Beckman Coulter, CA) and quantified by qPCR before and after amplification using NEB library quantification kit. Sequencing for this salivary gland RNA-Seq was performed on a NextSeq 500 (Illumina, CA).
Aleman J.R., Kuhn T.M., Pascual-Garcia P., Gospocic J., Lan Y., Bonasio R., Little S.C, & Capelson M. (2021). Correct dosage of X chromosome transcription is controlled by a nuclear pore component. Cell reports, 35(11), 109236.
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7

RNA-Seq of Harpegnathos Brains

Cited 1 time
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Harpegnathos brains without optic lobes (referred to as “brains” in the text) were dissected from single individuals and homogenized in TRIzol (Thermo Fisher Scientific, MA). RNA was purified and its quality visualized on agarose-formaldehyde gels. Typical yields amounted to 1.2 μg total RNA per brain. For RT-qPCR, 7 ng were assayed per 10 μl reaction using the RNA-to-Ct single-step kit (Thermo Fisher). The RNA for Rpl32, encoding a ribosomal protein, was used as a normalization control.
For library preparation, polyA+ RNA was isolated from 500 ng total RNA using Dynabeads Oligo(dT)25 (Thermo Fisher) beads and constructed into strand-specific libraries using the dUTP method (Parkhomchuk et al., 2009 (link)). UTP-marked cDNA was end-repaired using end-repair mix (Enzymatics, MA), tailed with deoxyadenine using Klenow exo− (Enzymatics), and ligated to custom dual-indexed adapters with T4 DNA ligase (Enzymatics). Libraries were size-selected with SPRIselect beads (Beckman Coulter, CA) and quantified by qPCR before and after amplification. Sequencing for Fig. 1 (d120 transition) and Fig. 5 (peptide and siRNA injections) was performed on a NextSeq 500 (Illumina, CA). Sequencing for Fig. 2E (Monomorium pharaonis) was performed on a HiSeq 2500 (Illumina). Sequencing for Fig. 3A and Fig. 6A (transition time points) was performed on a HiSeq2000 (Illumina).
Gospocic J., Shields E.J., Glastad K.M., Lin Y., Penick C.A., Yan H., Mikheyev A.S., Linksvayer T.A., Garcia B.A., Berger S.L., Liebig J., Reinberg D, & Bonasio R. (2017). The neuropeptide corazonin controls social behavior and caste identity in ants. Cell, 170(4), 748-759.e12.
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8

CRISPR-Cas9 Genome Editing Workflow

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle Medium DMEM (Gibco), fetal bovine serum (Gibco), Turbofect, TranscriptAid T7 High Yield Transcription Kit, Qubit dsDNA HS Assay Kit, Taq DNA polymerase (recombinant), Platinum Taq DNA polymerase, 0.45 µm sterile filters and penicillin/streptomycin were purchased from Thermo Fischer Scientific, protease inhibitor cocktail was purchased from Roche Diagnostics. DNA oligonucleotides, trimethoprim (TMP), chloroquine, polybrene, puromycin, calcium-phosphate and GenElute HP Plasmid Miniprep kit were acquired from Sigma-Aldrich. ZymoPure Plasmid Midiprep kit and RNA Clean & Concentrator kit were purchased from Zymo Research. NEBuilder HiFi DNA Assembly Master Mix and Q5 High-Fidelity DNA Polymerase were obtained from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit was purchased from Macherey-Nagel. Two millimeter electroporation cuvettes was acquired from Cell Projects Ltd, SF Cell Line 4D-Nucleofector X Kit S were purchased from Lonza, Bioruptor 0.5 ml Microtubes for DNA Shearing from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA universal qPCR Master Mix was purchased from KAPA Biosystems.
Kulcsár P.I., Tálas A., Ligeti Z., Tóth E., Rakvács Z., Bartos Z., Krausz S.L., Welker Á., Végi V.L., Huszár K, & Welker E. (2023). A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets. Nature Communications, 14, 5746.
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9

Molecular Cloning and Transfection Protocol

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle Medium DMEM (Gibco), fetal bovine serum (Gibco), Turbofect, Qubit dsDNA HS Assay Kit, Taq DNA polymerase (recombinant), TranscriptAid T7 High Yield Transcription Kit, Platinum Taq DNA polymerase, 0.45 µm sterile filters and penicillin/streptomycin were purchased from Thermo Fischer Scientific. DNA oligonucleotides, trimethoprim (TMP) and GenElute HP Plasmid Miniprep kit were acquired from Sigma–Aldrich. ZymoPure Plasmid Midiprep, RNA Clean & Concentrator kit and Maxiprep kits were purchased from Zymo Research. NEBuilder HiFi DNA Assembly Master Mix and Q5 High-Fidelity DNA Polymerase were obtained from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit was purchased from Macherey-Nagel. SF Cell Line 4D-Nucleofector X Kit S were purchased from Lonza, Bioruptor 0.5 ml Microtubes for DNA Shearing from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA universal qPCR Master Mix was purchased from KAPA Biosystems.
Kulcsár P.I., Tálas A., Ligeti Z., Krausz S.L, & Welker E. (2022). SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e. Nature Communications, 13, 6858.
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10

Molecular Biology Reagents and Protocols

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), Turbofect, Geltrex, StemPro Accutase, Qubit dsDNA HS Assay Kit, Platinum Taq DNA polymerase, and penicillin/streptomycin were purchased from Thermo Fischer Scientific Inc SF and SE Cell Line 4D-Nucleofector X Kit S were purchased from Lonza. Bioruptor 0.5 ml microtubes for DNA Shearing were from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA Universal qPCR Master Mix was purchased from KAPA Biosystems. DNA oligonucleotides and the GenElute HP Plasmid Miniprep and Midiprep kit used for plasmid purifications were acquired from Sigma-Aldrich. Q5 High-Fidelity DNA Polymerase, NEB5-alpha competent cells, and HiFi Assembly Master Mix were purchased from New England Biolabs Inc.
Simon D.A., Tálas A., Kulcsár P.I., Biczók Z., Krausz S.L., Várady G, & Welker E. (2022). PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells. eLife, 11, e69504.
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