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.
Klenow exonuclease minus
Manufactured by Qiagen
Klenow exonuclease minus is a laboratory enzyme used for DNA manipulation. It is a modified form of the Klenow fragment of DNA polymerase I, which lacks the 3'→5' exonuclease activity. This enzyme is useful for a variety of DNA-related applications, such as DNA labeling, nick translation, and fill-in reactions.
Automatically generated - may contain errors
Lab products found in correlation
End repair mix, by Qiagen (3 mentions)
Q5 dna polymerase, by New England Biolabs (3 mentions)
Minelute column, by Qiagen (3 mentions)
T4 dna ligase, by Qiagen (3 mentions)
Ampure xp, by Beckman Coulter (3 mentions)
Ultra 2 directional rna library prep kit, by New England Biolabs (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Fastselect rrna hmr, by Qiagen (2 mentions)
Uracil dna glycosylase, by Qiagen (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
3 protocols using klenow exonuclease minus
1
Chromatin Profiling and Transcriptomics in mESCs
2
DNA Library Preparation for Illumina Sequencing
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DNA 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 adaptors (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 dual-index barcode primers for Illumina sequencing (NEB E7600) using Q5 DNA polymerase (NEB M0491) and purified with MinElute. Uracil DNA glycosylase (Enzymatics) was added to the PCR amplification mix to degrade dUTP-containing molecules and remove adaptor hairpins. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38 × 2 paired-end cycles.
3
RNA-seq from mESCs using Trizol and rRNA depletion
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RNA samples were extracted from mESCs (n = 3 biological replicates) using Trizol reagent (Invitrogen) and subjected to DNase digestion with Turbo DNase (Ambion AM2238). RNA samples were then rRNAdepleted using FastSelect -rRNA HMR (Qiagen) and converted to cDNA using Ultra II Directional RNA Library Prep Kit (NEB E7760).
Library preparation and sequencing DNA 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 adaptors (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 dual index barcode primers for Illumina sequencing (NEB E7600) using Q5 DNA polymerase (NEB M0491) and purified with MinElute. Uracil DNA glycosylase (Enzymatics) was added to the PCR amplification mix to degrade dUTP-containing molecules and remove adaptor hairpins. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38x2 paired-end cycles.
Library preparation and sequencing DNA 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 adaptors (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 dual index barcode primers for Illumina sequencing (NEB E7600) using Q5 DNA polymerase (NEB M0491) and purified with MinElute. Uracil DNA glycosylase (Enzymatics) was added to the PCR amplification mix to degrade dUTP-containing molecules and remove adaptor hairpins. Sequencing was performed on a NextSeq 500 instrument (Illumina) with 38x2 paired-end cycles.
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