ChIP was performed with a commercial kit (SimpleChIP Enzymatic Chromatin IP Kit; Cell Signaling Technology, Danvers, MA, USA) in accordance with the manufacturer’s procedure. After de-crosslinking and proteinase K treatment, DNA was purified using phenol–chloroform extraction and ethanol precipitation with the co-precipitation reagent, Pellet Paint (Merck). For qPCR, see the RT-qPCR section. The primer sequences used in ChIP-qPCR assays are listed in Supplemental Table S2 . The following antibody was used: α-H3K36me3 (CMA333; a gift from Dr. Naohito Nozaki, MAB Institute, Inc.).
Pellet paint
Manufactured by Merck Group
Sourced in Germany
Pellet Paint is a laboratory equipment product designed for the application and deposition of various coatings and materials in a controlled and precise manner. It is a versatile tool used in research, development, and manufacturing settings across multiple industries.
Automatically generated - may contain errors
Lab products found in correlation
Rlt buffer, by Qiagen (2 mentions)
Qubit rna high sensitivity assay, by Thermo Fisher Scientific (2 mentions)
Trifast, by Avantor (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Glycoblue coprecipitant, by Thermo Fisher Scientific (1 mentions)
Rna solv, by Avantor (1 mentions)
Kod hot start dna polymerase, by Merck Group (1 mentions)
Superscript 3 rt kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Phenol chloroform, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Hiseq 3000 sequencer, by Illumina (1 mentions)
Elution buffer, by Qiagen (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Nhei hf, by New England Biolabs (1 mentions)
Gene pulser xcell, by Bio-Rad (1 mentions)
Magna nuclear rip cross linked nuclear rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
11 protocols using pellet paint
1
ChIP-qPCR Protocol for H3K36me3
2
Concentrating gDNA from Challenging Samples
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gDNA isolated from samples from which strain isolation was not possible (frozen canid heart tissue), unsuccessful, or not attempted was concentrated up to 20-fold by Na-acetate/ethanol precipitation. Briefly, 2 µL of a co-precipitant, PelletPaint (Merck-Millipore, Darmstadt, Germany), was added to each volume (20–100 µL) of gDNA to be concentrated along with 0.1 volumes of 3M Na-acetate (Merck-Millipore, Darmstadt, Germany) and mixed well. Next, two volumes of 100% ethanol were added to the sample and after a brief incubation at room temperature, the sample was centrifuged at top speed (16,000× g) in a tabletop centrifuge 5415R (Eppendorf, Hamburg, Germany) for 5 min. Next, the supernatant was discarded, the pellet was washed with 300 µL of 70% ethanol twice, and finally with 100% ethanol, and air-dried for 5–10 min. The pellet was re-suspended in a small volume of RNAse/DNAse free water.
3
RNA Extraction from Cultured Cells
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After removal of the cell culture supernatant, 500 µl of TriFast (Peqlab) or RNA-solv (VWR) were added to the dish. Cells were detached via gentle pipetting and transferred to RNAse-free microcentrifuge tubes. The tubes were vortexed for 15 s at maximum speed and stored at -80°C until RNA extraction. RNA extraction was performed according to the manufacturer’s protocol (Peqgold TriFast™ or VWR RNA-solv®). To maximize RNA yield, 2 µl of Pelletpaint (Merck Millipore) or 1.5 µl Glycoblue coprecipitant (Thermo Fisher) were added to the samples in the isopropanol precipitation step. A switch to RNA-solv and Glycoblue coprecipitant was necessary due to a shortage of supply and discontinuation of TriFast.
4
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from tumors using standard phenol/chloroform protocols and precipitated with isopropanol in the presence of Pellet Paint (Merck). The amplification was done using 2 ng of DNA and KOD hot start DNA polymerase (Merck). PCR fragments were sequenced by Eurofins Genomics. Primers used for PCR and sequencing are listed in Tables S1 and S2 .
5
Skin Biopsy RNA Extraction and cDNA Synthesis
6
EV RNA Extraction and Purification
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The EVs (1 × 108 EVs extracted from 10 ml of conditioned medium)were sorted into 100 µl of RLT buffer (Qiagen) and proceeded for RNA extraction. Briefly, 100 µl of RLT buffer with sorted EVs was mixed with 2 µl of pellet paint (Merck Millipore), vortexed briefly, 19 µl of 3 M Sodium Acetate (pH 5.5) and 300 µl of 100% ethanol was added and vortexed briefly and incubated at + 4 °C overnight. The contents were then centrifuged at 16,000 g for 15 min at 4 °C and the supernatant wascarefully discarded without disturbing the pellet. The pellet was washed twice with fresh 1 ml of 80% ethanol and air dried. The pellet was resuspended in 10 µl of RNase free water and stored at -80 °C till further use.
7
Genomic DNA Extraction from Tissues and Cells
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Tissue homogenates and cell line pellets were resuspended in 500 μL of Lysis Buffer (50 mM Tris-HCl ph = 8.0, 200 mM NaCl, 10 mM EDTA, 1% SDS) containing 0.2 mg/ml Proteinase K (Sigma) and incubated O/N at 55ºC. For mouse genotyping, gDNA was extracted from a small fragment of the tail cut when the animal was 3-4 weeks old. In the case of peripheral blood, red blood cells were eliminated by incubating the samples with 1 ml of ACK Lysis Buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA, pH 7.2-7.4) for 5 min at RT. Then, samples were centrifuged at 1,500 g for 5 min and the remaining cells were resuspended in Lysis Buffer with Proteinase K as described. On the following day, 500 μL Phenol-Chloroform (Sigma) was added to each sample, and then centrifuged at 9,500 g at RT for 10 min. Chloroform was added to the aqueous phase and samples were centrifuged again at 9,500 g at RT for another 10 min.
Afterwards, 1 mL of absolute ethanol along with 1 μL Pellet Paint (Merck) were added to the samples. Then, they were centrifuged at 9,500 g at 4ºC for 10 min. The resulting pellet was washed with 70% ethanol and the samples were once again centrifuged at 9,500 g and 4ºC for 10 min. Finally, gDNA was eluted in 8 mM NaOH, and the pH was adjusted with HEPES. After an O/N incubation at 4ºC, all gDNA samples were sonicated before measuring their concentration.
Afterwards, 1 mL of absolute ethanol along with 1 μL Pellet Paint (Merck) were added to the samples. Then, they were centrifuged at 9,500 g at 4ºC for 10 min. The resulting pellet was washed with 70% ethanol and the samples were once again centrifuged at 9,500 g and 4ºC for 10 min. Finally, gDNA was eluted in 8 mM NaOH, and the pH was adjusted with HEPES. After an O/N incubation at 4ºC, all gDNA samples were sonicated before measuring their concentration.
8
RNA Extraction and Bulk RNA-seq Protocol
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To extract bulk RNA from cells, the pellets were resuspended in 500 μl of TRI Reagent. After 5 min, 100 μl of chloroform was added and the tubes were shaken by hand for one minute. After 15 min incubation, the samples were centrifuged at 12 000 × g for 15 min at 4°C. 300 μl of the aqueous phase were then mixed thoroughly with 300 μl of Isopropanol, 30 μl of 3M Sodium Acetate and 1 μl of Pellet Paint (Merck 69049) and incubated over night at −20°C. The following day, the samples were centrifuged at 20 000 × g g for 30 min and the pellets washed twice with 600 μl of 70% ethanol. After drying, the pellets were resuspended in 15 μl of Elution Buffer and the concentration of RNA was measured using the Qubit RNA High Sensitivity Assay (Thermo Fischer Q32852) according to the manufacturer's instructions.
After diluting the samples, 2 ng of RNA were used as input for the Smart-seq2 RNA-sequencing protocol (41 (link)) and 50 bp single ends were sequenced on an Illumina HiSeq 3000 sequencer. Reads were mapped to the ENSEMBL human transcriptome GRCh37 using Tophat 2.1.1 to generate the read count matrix.
After diluting the samples, 2 ng of RNA were used as input for the Smart-seq2 RNA-sequencing protocol (41 (link)) and 50 bp single ends were sequenced on an Illumina HiSeq 3000 sequencer. Reads were mapped to the ENSEMBL human transcriptome GRCh37 using Tophat 2.1.1 to generate the read count matrix.
9
Extracting and Sequencing Cellular RNA
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Cell or EV RNA was extracted (32) and precipitated as previously described (3) by incubating 500 μl of TRI reagent (Sigma-Aldrich), adding 100 μl of chloroform and shaking vigorously. After a 15-min incubation, samples were centrifuged at 12,000g for 15 min at 4°C and 300 μl of aqueous phase was mixed with 300 μl of isopropanol, 30 μl of 3 M sodium acetate, and 1 μl of pellet paint (Merck) and incubated over night at -20°C. The next morning, samples were centrifuged at 20,000g for 30 min at 4°C, the pellets were washed two times with 700 μl of 70% ethanol, before drying and resuspending in 15 μl of elution buffer (Qiagen). RNA concentrations were measured using Qubit RNA high-sensitivity assay (Thermo Fisher Scientific) and 2 ng was used to generate full-length complementary DNA by Smart-seq2, which uses an oligo dT primer (23) . Fifty-base pair single-end reads were sequenced on a HiSeq3000 (Illumina), converted to fastq using bcl2fastq, adapters trimmed using Trim Galore, and the resulting reads aligned to the ENSEMBL human transcriptome GRCh37 or ENSEMBL mouse transcriptome GRCm39 using Tophat 2.1.1. To generate the normalized count matrix, DEseqDataSetFromMatrix and estimateSizeFactors were applied from the DEseq2 package in R (30) .
10
Construction of sc-(V1V2)³ Yeast Display Library
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The sc-(V1V2)3 library was prepared essentially as described previously (Chao et al., 2006 (link); Grimm et al., 2015 (link)). Inserts for an sc-(V1V2)3 yeast display library were constructed by error-prone PCR (epPCR) as previously described (Fromant et al., 1995 (link)), aiming for four amino acid mutations per gene. Products from this epPCR reaction were purified (Geneaid) and subjected to a second round of standard amplification using Phusion polymerase. Vector (250 μg of pCT) was prepared by restricting with BamHI-HF and NheI-HF (NEB). Vector was gel extracted using a DNA Extraction Maxi Kit (Geneaid), and both insert and vector were ethanol-precipitated with Pellet Paint (Millipore), and resuspended in water. A total of 600 μL of electrocompetent S. cerevisiae EBY100 were prepared as described previously (Chao et al., 2006 (link)), mixed with all insert and vector, and split into four 2 mm cuvettes. Cells were transformed at 1.2 kV and 25 μF using a Gene Pulser Xcell (Bio-Rad), recovered in a 1:1 mixture of 1 M sorbitol:YPD at 30°C for 1 h. Cells were transferred to 1 L SDCAA media, and serial dilutions were plated on SDCAA plates for 2 days at 30°C to estimate library diversity.
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