Half a million Hela-FRT cells expressing either UTX long or Δ14 isoforms as GFP fusions were harvested and washed twice in cold PBS. The cells were immobilized on concanavalin A-conjugated paramagnetic beads, permeabilized with 0.05% digitonin and subjected to the greenCUT&RUN protocol, as described before [32 (link)]. We added mononucleosomal Drosophila DNA as spike-in DNA for normalization purposes. Sequencing libraries were prepared using the NEB Next Ultra II kit (New England Biolabs, Ipswich, MA, USA). The resulting DNA quantity and size distribution was assessed using a Qubit instrument (Invitrogen, Waltham, MA, USA) and Agilent Bioanalyzer chips (DNA high sensitivity assay), respectively.
Agilent bioanalyzer chips
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Bioanalyzer chips are microfluidic devices designed for the analysis of biological samples. They provide automated, sensitive, and efficient separation and detection of DNA, RNA, and proteins using electrophoresis technology.
Automatically generated - may contain errors
Lab products found in correlation
Qubit instrument, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 kit, by New England Biolabs (1 mentions)
Puregene dna isolation kit, by Qiagen (1 mentions)
Hiseq 2500 sequencing platform, by Illumina (1 mentions)
Ambion mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Nebnext ultra 2, by New England Biolabs (1 mentions)
Neb multiplex oligo set 1 2, by New England Biolabs (1 mentions)
4 protocols using agilent bioanalyzer chips
1
greenCUT&RUN for UTX Isoforms
2
Methylation Profiling of Hippocampal DNA
3
Transcriptome Analysis of Oomycete Pathogens
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Ph. colocasiae, Ph. cactorum, Ph. plurivora, Py. oligandrum, and Py. periplocum were individually cultured in liquid V8 juice media and L. giganteum on PYG liquid medium (Domnas et al., 1977 (link)) for 3 days at 20°C and collected by gravity filtration. The different culture samples were snap frozen in liquid nitrogen and kept at −70°C until RNA extraction. Total RNA was obtained using the Ambion mirVana miRNA isolation kit according to the manufacturer’s instructions (Invitrogen, Waltham, MA), and RNA quality was assessed using Agilent Bioanalyzer chips (Agilent Technologies, Santa Clara, CA). The sequencing was carried out at the SciLife sequencing facility in Stockholm using an Illumina HiSeq2500 sequencing platform in High Output mode, SR 1x50bp. FastQC v0.11.3 (Andrews, 2010 ) was used to examine the quality of raw reads.
4
Genome-Wide Localization of Menin and JunD
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Genome localization analysis of GFP-tagged menin wild type, R52G, E255K, E359K or E408Q and GFP-tagged JunD was performed by greenCUT&RUN with the combination of enhancer-MNase and LaG16-MNase as described [33 (link)]. To localize MLL1 or RNA polymerase II standard CUT&RUN [32 (link)], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 µg/ml. For standard CUT&RUN and greenCUT&RUN mononucleosomal Drosophila DNA was used as spike-in DNA for normalization purposes and sequencing libraries were prepared as described (Nizamuddin et al., 2021). In brief, purified DNA fragments were subjected to library preparation with NEB Next Ultra II and NEB Multiplex Oligo Set I/II as per manufacturer (New England Biolabs) protocol without size selection. For each library, DNA concentration was determined using a Qubit instrument (Invitrogen, USA) and size distribution was analyzed with Agilent Bioanalyzer chips (DNA high sensitivity assay). The 75-nucleotide paired-end sequencing reads were generated (Illumina, HiSeq 3000) with 6–32 M reads per sample (Additional file 6 : Table S2). These NGS data have been deposited to Sequence Read Archive (52) under the accession number PRJNA772915.
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