Total RNA was extracted from sorted Treg cells using AllPrep DNA/RNA Micro Kit (Qiagen, Valencia CA) following manufacture’s protocol. RNA quality was assessed by a 2100 BioAnalyzer (Agilent Technologies, Santa Clara CA) at the University of Rochester Genomic Research Center. Total RNA from each sample was used for cDNA synthesis using the Ovation® RNA-Seq, with normalized RNA input for each sample (NuGEN, San Carlos CA). Amplified cDNA product was purified through a MinElute reaction cleanup column (Qiagen). Amplified cDNA quality was determined with a 2100 BioAnalyzer (Agilent Technologies).
Minelute reaction cleanup column
Manufactured by Qiagen
Sourced in Germany
The MinElute Reaction Cleanup columns are designed for the purification of DNA fragments from enzymatic reactions, such as PCR, sequencing, or restriction digests. The columns efficiently remove unwanted reaction components, including primers, dNTPs, enzymes, and salts, allowing for the recovery of high-quality DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Protein g agarose prepacked columns, by Active Motif (2 mentions)
Chip it high sensitivity kit, by Active Motif (2 mentions)
Allprep dna rna micro kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Histone h3 antibody, by Cell Signaling Technology (1 mentions)
Bioruptor pico instrument, by Diagenode (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
5 protocols using minelute reaction cleanup column
1
Treg Cell RNA Extraction and Profiling
2
Plasmid Assembly and Transformation
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100 ng vector and 12 ng insert were assembled in a total reaction volume of 100 μl (NEBuilder® HiFi DNA Assembly Master Mix, NEB). The reaction was cleaned via a Minelute reaction cleanup column (Qiagen) and transformed into 6 × 50 μl electrocompetent E. coli (Endura™ ElectroCompetent Cells, Lucigen) using a 1.0 mm cuvette, 25 μF, 400 Ohms, 1,800 Volts. Bacteria were plated on several 24 × 24 cm agar plates, and colonies were grown overnight at 30°C. Colonies were scraped into LB medium, and the contained plasmids were isolated by Maxiprep.
3
ChIP-Seq Assay for Hepatocyte Chromatin
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Chromatin was prepared from hepatocytes for ChIP assays as previously described90 (link). Cells were fixed with 4% paraformaldehyde for 15 min, then glycine was added to a final concentration of 0.125 M and incubated for 10 min before harvesting. Chromatin was sonicated using a Bioruptor Pico (Diagenode, Denville, NJ, USA). Chromatin preparations were subjected to ChIP using a ChIP-IT High Sensitivity Kit and Protein G Agarose Prepacked Columns (Active Motif, Carlsbad, CA, USA) using either PPARα (Abcam Ab24509) antibody, normal rabbit IgG (Cell Signaling Technologies #2729S), or Histone H3 (Cell Signaling Technologies #4620) antibody. Rabbit IgG and Histone H3 were used as negative and positive controls, respectively. DNA was purified and concentrated using MinElute Reaction Cleanup columns (Qiagen). qRT-PCR and conventional PCR were performed using 2 μl of ChIP DNA samples from the 50 μl of purified samples using gene-specific primers (Supplementary Table 7 ). Cycle threshold (Ct) values of ChIP and input samples were calculated and presented as fold change.
4
ChIP Assay for Hepatocyte Chromatin
5
Synthesis and Purification of Capped cRNA
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To prepare templates for cRNA synthesis, plasmids were linearized with EcoRI-HF (pNKS2) or NotI-HF (pUC19o) from New England Biolabs GmbH (Frankfurt am Main, Germany) and purified via MinElute Reaction Cleanup columns (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Alternatively, templates (including the 5’-terminal RNA polymerase promoter site (T7 or SP6) and the 3’-terminal poly A) were amplified by PCR and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol.
Capped cRNA was synthesized using the mMESSAGE mMACHINE SP6 or T7 Transcription Kits (Invitrogen/Thermo Fisher Scientific Inc, Schwerte, Germany), precipitated with LiCl, and dissolved in nuclease-free water (1 µg/µl if not stated otherwise).
The amber suppressor tRNA sequence was translated from the plasmid pANAP (Addgene #48696) (Chatterjee et al., 2013 (link)), provided with an universal 3’-terminal CCA-sequence (important for tRNA aminoacylation and translation), and chemically synthesized and purified via PAGE and HPLC (biomers.net GmbH, Ulm, Germany).
Capped cRNA was synthesized using the mMESSAGE mMACHINE SP6 or T7 Transcription Kits (Invitrogen/Thermo Fisher Scientific Inc, Schwerte, Germany), precipitated with LiCl, and dissolved in nuclease-free water (1 µg/µl if not stated otherwise).
The amber suppressor tRNA sequence was translated from the plasmid pANAP (Addgene #48696) (Chatterjee et al., 2013 (link)), provided with an universal 3’-terminal CCA-sequence (important for tRNA aminoacylation and translation), and chemically synthesized and purified via PAGE and HPLC (biomers.net GmbH, Ulm, Germany).
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