Two thousand Hydra polyps were crosslinked with 1% formaldehyde, lysed and sonicated in sonication buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 1% SDS, 4% NP-40, 1 mM PMSF) to obtain an average chromatin size of 300 bp. Chromatin was pre-cleared using 50 μL of a 50% protein A sepharose (GE healthcare) slurry for 1 h at 4°C with gentle inverting. Immunoprecipitations were carried out in 1 ml of ChIP buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton-X 100). Input chromatin was obtained after preclearing, by de-crosslinking and purifying input DNA using a Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4°C for 14–16 h. The samples were then incubated with 50 μL of a 50% protein A sepharose slurry for 3 h at 4°C with gentle inverting. ChIP samples were reverse-crosslinked and the DNA was purified using a Qiaquick column (Qiagen). Q-PCR using SYBR green was used to validate known target sites before and after sequencing.
Qiaquick column
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom
Qiaquick columns are a type of laboratory equipment used for DNA purification. They are designed to efficiently capture and purify DNA from various sample types, such as PCR reactions, enzymatic digests, or gel extractions. The columns contain a silica-based membrane that selectively binds DNA, allowing for the removal of contaminants and salts during the purification process.
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Lab products found in correlation
Hiseq 2500, by Illumina (4 mentions)
Thruplex fd prep kit, by Takara Bio (4 mentions)
H3k27ac antibody, by Diagenode (4 mentions)
Protein a and protein g beads, by Thermo Fisher Scientific (4 mentions)
E210 sonicator, by Covaris (4 mentions)
Eb buffer, by Qiagen (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Poly di dc, by Merck Group (2 mentions)
Micro bio spin column, by Bio-Rad (2 mentions)
Q sepharose resin slurry, by GE Healthcare (2 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Dnase 1, by Promega (2 mentions)
Fuji medical x ray film, by Fujifilm (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
105 protocols using qiaquick column
1
Chromatin Immunoprecipitation in Hydra
2
Hydra ChIP-seq Protocol
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Two thousand Hydra polyps each for DMSO control and ALP treatment were cross-linked with 1% formaldehyde, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1% SDS, 4% NP-40, 1 mM PMSF) to obtain an average chromatin size of 300 bp. Chromatin was pre-cleared using 50 μl of a 50% protein A sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of ChIP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton-X 100) with Anti-H3K4me3, Anti-H3K27ac, Anti-H3K9ac and Anti-H3. An appropriate IgG control was also used with inverting at 4 °C for 14–16 h. The samples were then incubated with 50 μl of a 50% Protein A sepharose slurry (saturated with 0.5% BSA and 10 mg/ml yeast tRNA) for 3 h at 4 °C with gentle inverting. ChIP samples were reverse-crosslinked, and the DNA was purified using a Qiaquick column (Qiagen). Input chromatin was obtained after preclearing, by de-crosslinking and purifying input DNA using a Qiaquick column (Qiagen) according to manufacturer’s instructions. Purified DNA was subjected to library preparation for sequencing or used for quantitative PCR. The fold enrichment was calculated using the formula—Fold enrichment = 2(Ct (Target antibody)−Ct (IgG)). The details of the primers used to perform the ChIP RT-PCR are provided in Additional file 9 : Table S8.
3
cDNA Normalization Protocol for Optimizing Transcriptome Analysis
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cDNA normalization was performed from total RNAs with MINT and TRIMMER kits from Evrogen according to the manufacturer’s instruction, except that the number of PCR cycles for material amplification was adapted to our material. First, full length double stranded (ds) cDNA were synthetized from 2 μg of total RNA using the MINT kit [55 (link)]. First strand was synthetized from a fusion primer containing an oligo (dT) stretch to anneal RNA polyA tails. A poly (dC) stretch was incorporated at the end of the first strand, and used for priming the synthesis of the second strand. Full length (ds) cDNA were subsequently amplified by PCR, purified on Qiaquick columns (Qiagen) and checked for quality and yield before normalization. Normalization was done with the TRIMMER kit (Evrogen) which is based on DSN technology [56 (link)]. The method involves denaturation-reassociation of cDNA, Duplex Specific Nuclease (DSN) degradation of the ds-fraction corresponding to abundant transcripts and PCR amplification of the single strand (ss) DNA fraction. We started from 600 ng (ds) cDNA for normalization and after denaturation, incubated samples at 68°C for five hours for renaturation. After degradation of (ds) complexes by DSN, we made two runs of PCR amplification for optimal recovery. Normalized cDNA was then purified on Qiaquick columns (Qiagen) and yield was measured by spectrophotometry.
4
Cultivation of Methanobrevibacter arboriphilus
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Methanobrevibacter arboriphilus strain DH1 (DSM 1125) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The methanogenic archaeon was cultivated under anaerobic conditions using a medium described by Asakawa et al. [12 ]. DNA was isolated from the enrichment culture by phenol-chloroform extraction followed by a QIAquick column (Qiagen, Hilden, Germany) purification [23 (link), 24 (link)].
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Methanobrevibacter arboriphilus strain DH1 (DSM 1125) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The methanogenic archaeon was cultivated under anaerobic conditions using a medium described by Asakawa et al. [12 ]. DNA was isolated from the enrichment culture by phenol-chloroform extraction followed by a QIAquick column (Qiagen, Hilden, Germany) purification [23 (link), 24 (link)].
5
ChIP Assay for NRF2 Transcription Factor
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ChIP experiments were performed using the ChIP Assay Kit (Beyotime, China, Cat No.P2078) according to the manufacturer's protocol. Cells were incubated for 10 min in PBS containing 1% formaldehyde and for 5 min in 0.125 M glycine. After centrifugation, cell pellets were resuspended in lysis buffer (1% SDS, 50 mM Tris·HCl pH 8, 10 mM EDTA). Sonication was performed with Bioruptor (Diagenode). Lysates were precleared with 30 μL of protein A-agarose, incubated with 10 μg of anti-NRF2 antibody (Abcam, CA, Cat No. ab62352) or rabbit IgG(Abcam, CA, Cat No. ab172730) overnight at 4°C on rotation, followed by incubation with 30 μL of protein A-agarose for 2 h. After washing the immune complexes were eluted from beads in a buffer containing 1% SDS and 0.1 M NaHCO3. Cross-linking was reversed overnight at 65°C, and DNA fragments were purified using the QIAquick column (Qiagen, Germany, Cat No. 28106). Quantitative PCRs were performed using 2 μL of DNA in triplicate.
6
Plasmid Preparation for Cell-free Reactions
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Example 44
Plasmids used in this study were constructed using standard cloning procedures and maintained in a KL740 strain if using an OR2-OR1 promoter (29° C.), a MG1655Z1 strain if using a Pl-tetO1 or Pl-lacO1 promoter, a BL21-DE3 strain for protein purification, a BL21 strain for promoter characterization, or a JM109 strain for all other constructs. KL740 upregulates a temperature sensitive lambda cI repressor, and MG1655Z1 upregulates tetR and lacI. PCR products were amplified using Pfu Phusion Polymerase (New England Biolabs) for all constructs except for those labeled with AlexaFluor-588-5-dUTP, which used Taq Polymerase (New England Biolabs), and were DpnI digested. Plasmids were either miniprepped using a PureYield column (Promega) or midiprepped using a NucleoBond Xtra Midi column (Macherey-Nagel). All plasmids were processed at stationery phase. Before use in the cell-free reaction, both plasmids and PCR products underwent an additional PCR purification step using a QiaQuick column (Qiagen), which removed excess salt detrimental to TX-TL, and were eluted and stored in 10 mM Tris-Cl solution, pH 8.5 at 4° C. for short-term storage and −20° C. for long-term storage.
7
Exome Sequencing Library Preparation
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Genomic DNA (3 μg) was first sheared into 200‐ to 300‐bp fragments using a Covaris S2 sonicator (Covaris, Inc., Woburn, MA) and then purified using a QiaQuick column (Qiagen, Chatsworth, CA). Illumina‐compatible sequencing libraries were prepared using Agilent library preparation reagents as per the manufacturer's instructions. A five‐cycle enrichment PCR was used to generate the libraries, which were captured using the Agilent v5 exome reagent hybridization probe set followed by ten cycles of posthybridization enrichment PCR. Paired‐end, 100‐bp reads were generated using a HiSeq 2500 in high‐output mode with five samples pooled per lane. The sequence data were aligned to the human genome (hg19) using BWA [Li and Durbin, 2009] and variants identified using GATKlite [McKenna et al., 2010 ]. Nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Similarly, amino acid numbering uses +1 at the initiating methionine residue of the reference sequence.
8
Footprinting of PurB-PhoP Regulatory Region
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DNA fragments corresponding to the 3′ end of the purB coding region with the purB-phoP intergenic region were generated by PCR from wild-type S. Typhimurium (14028s) using primers 14244 and 32P-labeled 14217. The DNA fragments were gel purified with QIAquick column (Qiagen). A total of ∼105 cpm of labeled DNA probe (∼2 nM) and purified SsrBc (0, 0.25 and 1 μM) were mixed with the same binding buffer used in the electrophoretic mobility shift assay including 50 ng μl−1 of poly(dI-dC) (Sigma) in a total volume of 20 μl and incubated for 15 min at room temperature. DNase I (Promega) (0.01 units), 10 mM CaCl2, and 10 mM MgCl2 were added and incubated for 3 min at room temperature. The reaction was stopped by the addition of 100 μl of phenol chloroform, and the aqueous phase was precipitated with ethanol. The precipitate was dissolved in sequence-loading buffer and electrophoresed on a 6% acrylamide/7 M urea gel together with a sequence ladder initiated with the labeled primer by using the T7 Sequenase 2.0 DNA-sequencing kit (Amersham Biosciences), and the gels were dried and autoradiographed (Fujifilm).
9
ATAC-Seq Protocol for Profiling Chromatin Accessibility
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In short, nuclei were isolated by adding 500 μl lysis buffer (10 mM Tris/HCl [pH 7.5], 10 mM NaCl, 3 mM MgCl2 and 0.2% [vol/vol] IGEPAL, in Milli-Q water/PBS [1:1]) to the cell pellet (iPSCs and fibroblasts) or to the cells on the plate (iNeurons), followed by mechanically dissociating the cells using a pipette. Nuclei were pelleted, washed, and incubated with the Tn5 transposase in a shaking heat block at 37°C/650 rpm for 1 h. The reaction was stopped by adding 5 μl clean-up buffer (0.9 M NaCl and 0.3 M EDTA [pH 8] in nuclease-free water), 2 μl proteinase K (10 mg/ml) and 2 μl 5% SDS. DNA was purified using normal-phase 2× AMPure bead purification (Beckman Coulter), PCR amplified for 8 PCR cycles, size-selected using reverse-phase 0.55× AMPure bead purification, purified using a Qiaquick column (Qiagen), PCR amplified for another eight PCR cycles, and again purified on column. Sequencing was performed on an Illumina NextSeq 500 using HighOutput kit v2 for 75 cycles (paired-end 2 × 43 bp). The number of mapped ATAC-seq reads can be found in Supplementary Table S3 .
10
Chromatin Immunoprecipitation and ChIP-qPCR
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Chromatin immunoprecipitation (ChIP) and ChIP-qPCR are performed as described previously [18 (link), 67 (link)]. Infected RBCs were cross-linked with 1% formaldehyde (Catalogue number- 28908, THERMO Scientific) for 10 min, lysed and sonicated. ChIP samples were reverse-crosslinked and DNA was purified using a Qiaquick column (Qiagen). Target sites obtained from ChIP-seq analysis were further validated by quantitative PCR using Power SYBR Green Master Mix (Applied Biosystems).
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