Genes with age-related epigenetic changes in humans and mice were identified through literature searches. The genes tested and the studies demonstrating their age-related methylation are given in Table1 . Candidate 5′ regulatory region sequences were taken from GenBank and used as queries for blastn (Altschul et al. 1990 (link)) searches of cetacean sequences in GenBank and BLAT searches of the dolphin (Tursiops truncatus) genome (Vollmer & Rosel 2012 (link)). Where candidate genes had a clearly orthologous 5′ regulatory regions in the T. truncatus genome, primers for amplification of humpback sequences were designed by eye based on all available homologous cetacean sequences. Humpback gene regions were amplified in 10 μL PCR reactions containing 5 μL 2× Phusion HF (NEB) master mix, 1 μm of each amplification primer, 10 ng of humpback whale DNA and milli-Q H20 with thermal cycling conditions appropriate to each primer set and predicted amplicon. The fragments were purified with Ampure magnetic beads (Agencourt) and bidirectionally sequenced by dye terminator v 3.1 chemistry on an ABI 3100 Sanger sequencer (Applied Biosystems).
Ampure magnetic beads
Manufactured by Beckman Coulter
Sourced in United States, Italy
AMPure magnetic beads are a product designed for purification of nucleic acids, including DNA and RNA, from various sample types. The beads utilize a paramagnetic particle technology to selectively bind nucleic acids, allowing for efficient removal of contaminants and impurities during the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (21 mentions)
Nextera xt fragmentation enzyme, by Illumina (18 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (13 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (10 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (8 mentions)
Nextera index kit, by Illumina (7 mentions)
Hiseq 4000, by Illumina (5 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (4 mentions)
Nextseq 550, by Illumina (3 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
Microplex library preparation kit v2, by Diagenode (3 mentions)
Hiseq 4000 platform, by Illumina (3 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (3 mentions)
Nextera xt kit, by Illumina (3 mentions)
Miseq reagent kit v3, by Illumina (3 mentions)
Hifi hotstart readymix, by Roche (2 mentions)
Miseq sequencing, by Illumina (2 mentions)
Adaptive focused acoustics machine, by Covaris (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Sm1323, by Thermo Fisher Scientific (2 mentions)
68 protocols using ampure magnetic beads
1
Cetacean Age-Related Epigenetics
2
Coral Microbial Community Analysis via 16S Amplicon Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the laboratory, coral mucus samples were thawed, centrifuged and supernatant decanted. DNA was purified using an organic extraction as previously described26 . After DNA extraction, microbial 16S amplicon libraries were generated using the primers 515F and 806R, both with added 454 sequencing adaptors and with Golay barcodes added to the reverse primer. Triplicate 25 μl reactions were conducted using the GoTaq Flexi system from Promega (Madison, WI, USA) with the following conditions per reaction: 1 × clear buffer, 1 mM dNTPs, 5 mM MgCl, 1 μM of each primer, 1u Taq polymerase and 1 μl of extracted DNA template. Thermocycling was conducted as follows: 1 cycle of 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 60 s and 72 °C for 90 s; and 1 cycle of 72 °C for 10 min. Amplification success was checked on a 1.5% agarose gel, and successful triplicate reactions were pooled and cleaned using AMPure magnetic beads from Agencourt. Before sequencing, libraries were quantified using a Qubit dsDNA HS kit from Invitrogen and then pooled into equimolar ratios. The pooled library was checked for amplicon length and purity on an Agilent Bioanalyzer 2100 and then sequenced on a 454 Roche pyrosequencer (GSJunior platform) at the Oregon State University's Center for Genome Research and Biocomputing Core Laboratories.
3
Amplification and Sequencing of Large Genomic Fragments
4
Precise RNA Template Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA templates were designed to include the 20-nt T7 RNA polymerase promoter sequence (5’-TTCTAATACGACTCACTATA-3’) followed by the remaining sequence encoding desired RNA. Double-stranded templates were prepared by extension of 60-nt DNA oligomers (IDT, Integrated DNA Technologies) with Phusion DNA polymerase (Finnzymes), using the following thermocycler protocol: denaturation for 30 sec at 98°C, 35 cycles of denaturation for 10 sec at 98°C annealing for 30 sec at 60 to 64°C , extension for 30 sec at 72°C; final extension for 10 min at 72 °C and cooling to 4°C.
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).
5
RNA-Seq Library Preparation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted using the Qiagen RNeasy Plant kit following the manufacturer’s protocol. The Illumina TruSeq RNA Sample Preparation Kit v2 was used for RNA library construction, using manufacturer suggested protocols, including Agencourt AMPure magnetic beads for cleaning steps. The 12 RNA libraries described above were barcoded with the TruSeq kit adapters, multiplexed 12 deep per lane, and sequenced across 3 lanes as technical replicates (Auer and Doerge 2010 (link)), resulting in a total of 36 individual RNA-Seq datasets. Sequencing reactions were conducted for 51 single-end cycles on the Illumina HiSequation 2000 at the Center for Genome Research and Biocomputing at Oregon State University.
6
TP53 Sequencing Panel Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TP53 sequencing was performed in all patients using a clinically validated 53-gene panel or a 28-gene panel assay. The 53-gene panel covers exons (codons) 2, 4–8, and 10 (1–12, 69–112, 126–253, 267–206, and 332–342) and the 28-gene panel covers exons (codons) 4–10 (41–224 and 234–367). Briefly, genomic DNA (gDNA) was extracted from bone marrow aspirate or peripheral blood of each case using an Autopure extractor (Qiagen, Valencia, CA, USA). A sequencing library was prepared using 250 ng of DNA template and either 53- or 28-gene panel. The sequencing library was purified using AMPure magnetic beads (Agencourt, Brea, CA, USA) and then subjected to MiSeq sequencer (Illumina Inc., San Diego, CA, USA) [24 (link)]. A minimum quality score of AQ30 is required for a minimum of 75% of bases sequenced ensuring high-quality sequencing results. Variant calling was performed with Illumina MiSeq Reporter Software 1.3.17 using human genome build 19 (hg 19) as a reference and sequencing reads were aligned using the Integrative Genomics Viewer (IGV, Broad Institute, MA, USA) [25 (link)]. For clinical reporting, a sequencing coverage of 250× (bi-directional true paired-end sequencing) and a variant frequency of 5% in a background of wild-type TP53 were used as cutoffs.
7
PCR Amplification of DNA Templates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA templates were designed to include the 20-nt T7 RNA polymerase promoter sequence (5′-TTCTAATACGACTCACTATA-3′) followed by the remaining sequence encoding desired RNA. Double-stranded templates were prepared by extension of 60-nt DNA oligomers (IDT, Integrated DNA Technologies) with Phusion DNA polymerase (Finnzymes), using the following thermocycler protocol: denaturation for 30 s at 98 °C, 35 cycles of denaturation for 10 s at 98 °C annealing for 30 s at 60–64 °C, extension for 30 s at 72 °C; final extension for 10 min at 72 °C and cooling to 4 °C.
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following the manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including the following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).
DNA samples were purified with AMPure magnetic beads (Agencourt, Beckman Coulter) following the manufacturer’s instructions. Sample concentrations were estimated based on UV absorbance at 260 nm measured on Nanodrop 100 or 8000 spectrophotometers. Verification of template length was accomplished by electrophoresis of all samples and 10-bp and 20-bp ladder length standards (Thermo Scientific O’RangeRuler SM1313 & SM1323) in 4% agarose gels (containing 0.5 mg/mL ethidium bromide) and 1x TBE (100 mM Tris, 83 mM boric acid, 1 mM disodium EDTA). All sample manipulations, including the following steps, were carried out in 96-well V-shaped polypropylene microplates (Greiner).
8
Amplification and Sequencing of Large Genomic Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time PCR was performed as described above using Kapa HiFi HotStart ReadyMix as directed by the manufacturer (Supplementary Table 7 : 5.8kb_forward/reverse, 5.8kb_f_alt/r_alt), depending on the desired target, and the following thermal cycling protocol: 95°C for 180 sec, repeat [98°C for 20 sec, 65°C for 15 sec, 72°C for 180 sec]. Samples were loaded on a 1% agarose gel and run for 1 hour at 100 V. Bands of the desired size were size selected and purified (Supplementary Fig. 9 ). A second, four-fold larger volume PCR using the same conditions was then run using the size-selected sample. The PCR products were pooled for each individual and purified using Agencourt AMPure magnetic beads, following the manufacturer’s protocol. Samples were eluted in 50 μl and loaded directly into the Covaris Adaptive Focused Acoustics machine. Standard shotgun libraries were then prepared for paired-end (2 × 250 bp) Illumina MiSeq sequencing (sequencing primers in Supplementary Table 7 : MiSeq_p7 and MiSeq_p5).
9
ChIP-seq Library Preparation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sequencing libraries were prepared from ~10 ng of ChIP and 1 μg of input DNA using NEBnext® kit (E6260, NEB) according to the manufacturer’s protocol. The PCR amplified library was purified using Agencourt AMPure magnetic beads for gel fragments size selected at 150–400 bp. Each ChIP and input library was sequenced from a single end to a length of 40 nucleotides on individual lanes of an Illumina Genome Analyzer IIx. Allele-specific read alignment and detailed bioinformatics analysis of data are outlined in Additional file 4 .
10
Illumina Library Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We fragmented genomic DNA to a target size of 300 bp on the Covaris M220. We prepared libraries using the NEBNext Ultra DNA Library Preparation Kit for Illumina and the Multiplex Oligos Kit for indexes (New England Biolabs), using 50–100 ng of genomic DNA input for each sample. The preparation involves end repair, dA tailing, adapter ligation, cleanup with Agencourt AMPure magnetic beads, PCR enrichment with incorporation of indexes and PCR cleanup. We quantified the final libraries using Qubit and library size was determined using the Agilent TapeStation 2200 as described by the manufacturer.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!