Satellite cells were isolated from adult Tg:Pax7-nGFP mice using in situ fixation to preserve Notch signalling from dissociation-induced downregulation39 . Cells were fixed as above in (2mM Di(N-succinimidyl) glutarate for 45min, followed by 10min with 1% formaldehyde at RT). Cell lysis and chromatin isolation were performed using Auto-TrueMicrochip kit (Diagenode, C01010140). Chromatin was sheared as above with 10 cycles of 30s on/off sonication using a Bioruptor Pico. 2x105 cells were used per ChIP and 2x103 per Input and IPs were performed using 2μl of anti-Rbpj antibody (Cell Signalling, 5313) or 0.5μl of rabbit control IgG antibody following the manufacturer guidelines. Immunoprecipitated chromatin preparations and input were purified using the Auto IPure kit v2 (Diagenode). RT-qPCR was performed using FastStart Universal SYBR Green Master mix (Roche, 04913914001) and analysis was performed using the 2-∆∆CT method38 (link) normalised to the Neg16 region. Primers used for ChIP-qPCR are listed in Supplementary Table 1 .
Auto ipure kit v2
Manufactured by Diagenode
The Auto IPure kit v2 is a laboratory device designed for automated DNA/RNA extraction and purification. Its core function is to facilitate the efficient and consistent isolation of nucleic acids from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor 200 ucd 300, by Diagenode (3 mentions)
Auto truemicrochip kit, by Diagenode (2 mentions)
Anti rbpj antibody, by Cell Signaling Technology (2 mentions)
Faststart universal sybr green master mix, by Roche (2 mentions)
Auto hmedip kit, by Diagenode (2 mentions)
Sx 8g ip star compact system, by Diagenode (2 mentions)
Nebnext ultra dna library preparation kit, by New England Biolabs (2 mentions)
Ip star compact automated system, by Diagenode (1 mentions)
Auto ideal chip seq kit for histones, by Diagenode (1 mentions)
Nebnext ultra directional rna library preparation kit, by New England Biolabs (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
6 protocols using auto ipure kit v2
1
Preservation of Notch Signaling in Satellite Cell Chromatin Immunoprecipitation
2
Preservation of Notch Signaling in Satellite Cell Chromatin Immunoprecipitation
3
Chromatin Immunoprecipitation Sequencing
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Cells were cross-linked in formaldehyde at a final concentration of 1% for 8 min. All experiments reagents were included in the Auto iDeal ChIP-seq kit for Histones (Diagenode, Liege, Belgium). Sonication was performed using a Bioruptor Plus sonication devise (Diagenode, Liege, Belgium) under the optimal conditions to shear cross-linked DNA to fragments if 100–300 base pairs in length. ChIP was conducted with the IPStar Compact Automated System (Diagenode, Liege, Belgium). ChIP were performed starting from 1 million cells per IP, using the indirect method in 200 μL final volume. Crossed-linked DNA was incubated 13 h with the antibody (H3K27me3 catalog number C1540195 or H3K4me3 catalog number C15410003) and 3 h with the beads. After 5 min washes, eluates were recovered and reverse cross-linked for 4 h at 65 °C. Samples were treated for 1 h with RNAse at 37 °C, prior to DNA purification with the Auto IPure kit v2 (Diagenode, C03010010). Libraries were performed using NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs). Sequencing was performed with Illumina NextSeq500 technology (Helixio, Clermont-Ferrand, France) using the following parameters: single-read, 50 bp, 40 million reads.
4
Methylation Profiling of Flower Pedicels
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Genomic DNA was isolated from WT and j-2 (LA3899) mutant flower pedicels as previously described and fragmentation was performed using Diagenode Bioruptor 200 UCD-300 (30 s then off 90 s for 25 cycles, low power position). Following steps were performed using Diagenode Auto hMeDIP KIT in the SX-8G IP-Star® Compact System. Anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode) was used for precipitation. DNA was then purified using Auto Ipure kit v2 (Diagenode). MeDIP-qPCR was performed by the same methods as the RT-qPCR using the purified immunoprecipitated DNAs as templates. Primers used for MeDIP-qPCR are listed in Supplementary Table 2 .
5
Methylation Profiling of Charmono Melon
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Genomic DNA was isolated from roots and leaves of the Charmono melon cultivar (Cucumis melo L. ssp. melo var cantalupensis) using E.Z.N.A Plant DNA Kit (Omega). Fragmentation was performed using Diagenode Bioruptor 200 UCD-300. MeDIP-Seq libraries construction was performed as described in Ruggieri et al. (2018) (link). Briefly, methylated DNA was precipitated with anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode), then purified using Auto Ipure kit v2 (Diagenode). Libraries were synthetized using NebNext Ultra DNA Library Preparation Kit (NEB) according to the manufacturer’s instructions and sequenced by Illumina Sequencing technology using the service of INRAE EPITRANS platform (Orsay, France).
6
Melon Methylome and Transcriptome Analysis
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Genomic DNA was isolated from roots and leaves of the cultivar Charentais (Cucumis melo L. subsp. melo var cantalupensis) using E.Z.N.A Plant DNA Kit (Omega). Fragmentation was performed using Diagenode Bioruptor 200 UCD-300 (30 s then off 90 s for 25 cycles, low power position). The following steps were performed using Diagenode Auto hMeDIP Kit in the SX-8G IP-Star® Compact System. Anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode) was used for precipitation. DNA was then purified using Auto Ipure kit v2 (Diagenode). Libraries were synthetized using NebNext Ultra DNA Library Preparation Kit (NEB) according to the manufacturer’s instructions and sequenced by Illumina technology. The same melon accession was used to collect the the total RNA from roots of 10 day old (cultivated in vitro) and from leaves of 3 weeks old, grown in a growth chamber (long day conditions, temperature: 27 °C (day) and 21 °C (night), relative humidity: 60%). The Nucleospin RNA kit (Macherey-Nagel) was used for the extraction according to the manufacturer’s instructions. Libraries were synthetized from 2 µg of total RNA using NEBNext Ultra Directional RNA library Preparation Kit (NEB) according to the manufacturer’s instructions. Two biological replicates were analysed for each tissue. Libraries were sequenced by Illumina technology.
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