ChIP was conducted using a ChIP Assay Kit (cat# 17-295, EMD Millipore, Billerica, MA, USA). After fixation with 1% formaldehyde for 10 min, the cells were subjected to decrosslinking with 0.125 M glycine for 5 min, washed with PBS, and lysed for 1 h on ice. The cell lysates were sonicated to generate chromatin fragments approximately 500 to 800 bp in length that were assessed by agarose gel electrophoresis. Following preclearing with Protein-A agarose, the samples were incubated with 5 μg of specific antibodies with rotation overnight at 4°C. Then, the immune complexes were precipitated with Protein-A agarose beads and sheared salmon sperm DNA, and the DNA fragments were purified using a QIAquick Spin Kit (Qiagen). The promoter segments containing a c-Fos binding site were amplified using PCR technology.
Qiaquick spin kit
Manufactured by Qiagen
Sourced in United States, Germany
The QIAquick Spin Kit is a lab equipment product designed for the purification of DNA fragments from enzymatic reactions or other sources. It utilizes a silica-membrane technology to efficiently capture and purify DNA samples, allowing for their subsequent use in downstream applications.
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Lab products found in correlation
Qiaamp dna ffpe tissue kit, by Qiagen (5 mentions)
Protein a agarose, by Roche (4 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Abi 3730 instrument, by Thermo Fisher Scientific (2 mentions)
Ab5131, by Abcam (2 mentions)
H 300, by Santa Cruz Biotechnology (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Dynal protein a, by Thermo Fisher Scientific (2 mentions)
Sc 2027, by Santa Cruz Biotechnology (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Bioruptor, by Diagenode (2 mentions)
H3k9ac, by Merck Group (2 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (2 mentions)
Lightcycler 480 system, by Roche (2 mentions)
Abi 3730, by Thermo Fisher Scientific (2 mentions)
Chip assay kit, by Merck Group (1 mentions)
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Abi prism 3130xl genetic analyzer system, by Thermo Fisher Scientific (1 mentions)
35 protocols using qiaquick spin kit
1
ChIP Assay for c-Fos Binding
2
Oncogene Mutation Analysis by PCR and Sanger Sequencing
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To investigate the mutation status of key oncogenes, we performed a polymerase chain reaction followed by Sanger sequencing. 43 Briefly, DNA from formalin-fixed paraffin-embedded tissue was extracted with a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). The tumor component of the slides was microdissected to increase the tumor cell ratio. Polymerase chain reaction products were purified using a QIAquick Spin Kit (Qiagen). Each purified product was directly sequenced using a forward primer with a BigDye Terminator version 3.1 cycle sequencing kit on an ABI 3730 instrument (Applied Biosystems Inc., Foster City, CA). A mutation analysis of HRAS (exons 2 and 3), AKT1 (exon 2), PIK3CA (exons 9 and 20), BRAF (exon 15), CTNNB1 (exon 3), and KRAS (exons 2 and 3) was performed. The primer sequences are listed in Supplementary Table S2 (Supplemental Digital Content 2, https://links.lww.com/PAS/A748).
3
Characterization of Oenococcus oeni Isolates
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All 250 isolates were observed with an optical microscope and those with coccal-shaped cells were selected for total DNA extraction according to Cappello et al. [18 (link)]. These isolates were assayed for O. oeni identification by 16S rDNA sequencing using primers and PCR cycling parameters previously described [18 (link)]. Amplicons were analyzed by agarose gel electrophoresis, purified using the QIAquick spin Kit (Qiagen Science Inc., Germantown, MD, USA), and sequenced at the Eurofins Genomics service (Eurofins Genomics, Edersberg, Germany) using the same primers employed for amplification. Sequence similarity searches were carried out using a basic local alignment search (BLAST) in the EMBL/GenBank databases. AFLP fingerprinting of all O. oeni isolated from Nero di Troia wine and ATCC BAA-1163, used as a reference strain, and clustering by the unweighted pair group method with arithmetic mean (UPGMA), were performed as previously described [18 (link)].
4
ChIP-PCR analysis of ELF5 in HC11 cells
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Approximately 5 × 106 cells of HC11 cells were subjected to ChIP assays, which were conducted using the EZ-ChIP Chromatin Immunoprecipitation Kit (EMD Millipore) according to the manufacturer’s manual. Five micrograms of antibodies were used for each pull-down assay. Immunoprecipitated DNA fragments were purified with the QIAquick Spin Kit (Qiagen) and analyzed by PCR. The primers are ELF5 forward (5′-TGCCTAGTGATACAGGGTCTCAT-3′) and ELF5 reverse (5′-CCAACACTCAGGCGGCAGAT-3′). Anti-RNA polymerase II antibody and primers are provided in the ChIP Kit as positive controls.
5
Cloning and Phylogenetic Analysis of RACE Sequences
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Cloning and sequence analysis. The RACE product bands were excised from the agarose gels and purified using the QIAquick Spin kit (Qiagen) according to the manufacturer's protocol. After purification, the RACE products were cloned into the pGEM T-Easy plasmid (Promega). Plasmids from positive colonies were analyzed by restriction; each clone was sequenced in both chains using universal primers and the Big Dye Terminator Ready Reaction kit (Perkin-Elmer) and analyzed in the ABI PRISM 3130xl Genetic Analyzer System (Applied Biosystems). Sequence information was analyzed using the CLC Bio Main Workbench (CLC Bio; Qiagen) as well as the BLAT (genome.ucsc.edu) and BLAST (blast.ncbi. nlm.nih.gov) algorithms.
In order to search for sequence similarity among clones, we performed a CLUSTAL alignment and checked it manually. Based on this alignment, we searched for the most adequate model of evolution using the FindModel server (http://www. hiv.lanl.gov/content/sequence/findmodel/findmodel.html), and the GTR model was selected to perform a maximum likelihood phylogenetic analysis employing a 1000-replicate bootstrap analysis. Finally, a tree was constructed using the neighborjoining method.
In order to search for sequence similarity among clones, we performed a CLUSTAL alignment and checked it manually. Based on this alignment, we searched for the most adequate model of evolution using the FindModel server (http://www. hiv.lanl.gov/content/sequence/findmodel/findmodel.html), and the GTR model was selected to perform a maximum likelihood phylogenetic analysis employing a 1000-replicate bootstrap analysis. Finally, a tree was constructed using the neighborjoining method.
6
Cloning and Sequencing of Regulatory Elements
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All amplified products obtained from secondary PCR were purified from agarose gel using a QIAquick Spin kit (Qiagen) and further cloned into the pTZ57R/T cloning vector system using TA cloning kit (Fermentas). Plasmids were isolated from different clones and were then processed for sequencing after purification. For removal of vector contamination, sequences were trimmed using VecScreen software (https://www.ncbi.nlm.nih.gov/tools/vecscreen/ ) and were aligned by BLASTn and ClustalW program. The presence of putative cis-regulatory elements were detected by PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html ) and Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html ) scan tools [13 (link)]).
7
Chromatin Immunoprecipitation Protocol
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Cells (5×106) were fixed at 37°C in RPMI with 1% formaldehyde for 10 min, lysed in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1 plus protease inhibitors and sonicated with a probe tip until DNA was an average size of 1 kilobase. Input was saved and lysate was diluted in immunoprecipitation buffer (1% Triton, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1) and mixed with beads (Dynal Protein A, Invitrogen) that were pre-bound overnight with antibodies to Nrf2 (H-300, Santa Cruz), ATF4 (11815, Cell Signaling), RNA pol II (pSer5, ab5131, abcam) or Rabbit IgG (sc-2027, Santa Cruz). Chromatin was immunoprecipitated overnight, and beads were washed six times with RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5 M LiCl) and twice with TE. Beads were incubated with 1% SDS, 0.1 M NaHCO3 for 30 min at room temperature, and then crosslinks were reversed on both the input and the immunoprecipitate by heating overnight in a 65°C water bath. DNA was purified with a QIAquick spin kit (Qiagen) and Q-PCR was performed in triplicate with a Fast Sybr green master mix on a Step One Real-Time PCR system (all Life Technologies). Primer sequences can be found in Supplementary Table 4 .
8
Chromatin Immunoprecipitation of p53 on miR-181a Promoter
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The NRVCs were washed with PBS and incubated with 1% formaldehyde for 10 min at room temperature. We quenched the cross-linking with 0.125 M glycine for 5 min and lysed cells for 1 h at 4 °C with 100 μL lysis buffer. We sonicated the cell lysates into chromatin fragments for 4 min 30 s with an average length of 500 to 800 base pairs (bp). The samples were precleared with Protein-A agarose (Roche), and 5 mg specific antibodies (antibody to p53 (SAB1306667, SIGMA) were added and rocked for overnight at 4 °C. Immunoprecipitates were captured with 10% (vol/vol) Protein-A agarose for 4 h and DNA fragments were purified with a QIAquick Spin Kit (Qiagen). We performed PCR with primers that encompassed p53 BS on the rat miR-181a promoter. The oligonucleotides were as follows: forward, 5′- CAGATGTCCATTTTTAGTC-3′; reverse, 5′- GTTTTTGAATCCCAAACT-3′.
9
ChIP Assay for Identifying ERRα Binding Sites
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ChIP assays were performed on MDA-MB-231 cells as previously described [20 (link)]. Briefly, cells were incubated for 10 min in phosphate-buffered saline containing 1% formaldehyde. After sonication with Bioruptor (Diagenode, Liège, Belgium), soluble chromatin fragments of 200 to 1000 bp in length were incubated with 5 μg of anti-hERRα antibody, rabbit anti-IgG antibody, or no antibody for 16 h at 4°C, followed by incubation with 80 μg of salmon sperm DNA/protein A-agarose for 2 h at 4°C. Immunoprecipitates were washed and eluted. Samples were then treated with RNase A (Roche Diagnostics) for 30 minutes at 37°C and with proteinase K (Roche Diagnostics) for 2 h at 42°C. Isolated DNA fragments were purified with QIAquick spin kit (Qiagen), and quantitative PCRs were performed using 2 μl of DNA in triplicate. The promoter of FN (−998 to +199) was scanned for putative ERREs using the MAPPER search engine [39 (link)]. The primers for four ERRE sites were listed at Supplementary Table S1 .
10
ChIP-qPCR Analysis of H3K9Ac
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Chromatin immunoprecipitation followed by RT-qPCR was performed as previously described38 with H3K9Ac (Millipore, 07-352). In brief, SCC13 cells were crosslinked with 1% formaldehyde/PBS for 15miin at RT, followed by quenching with 0.125M glycine. After washing twice with PBS, cells were collected in RIPA buffer, and then were sonicated to generate DNA fragments of approximately 500 bp in size. About 200ug of pre-cleared protein extract was used for immunoprecipitation overnight (>12hr) at 4 °C using protein A/G agarose beads (Santa Cruz, sc2003). Washed samples were eluted by incubation at 65 °C for 10 min with 1% SDS, and crosslinking was reversed by incubation at 65 °C for 6 hr with 200mM NaCl. DNA was purified using the QIAquick spin kit (Qiagen) and assessed by real-time qPCR using the LightCycler 480 system (Roche). The primers’ sequences are listed in Supplementary Table 9 .
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