The Mayo Clinic Specialized Program of Research Excellence (SPORE) in Pancreatic Cancer identified 14 clinically and histologically confirmed PDAC patients, who provided consent for use of tissue for research, and for whom frozen PDAC and adjacent pancreatic intraepithelial neoplasia (PanIN) tissues were available. LCM was used to individually isolate tumor, PanIN, or histologically normal cells from fresh frozen tissue sections and DNA was amplified directly by a single-step procedure using the Qiagen Repli-g WGA kit, as previously described (8 (link)–11 (link)). Mate pair sequencing (MPseq) libraries were assembled from WGA DNA, as previously published (9 (link)–11 (link)) using the Illumina MP kit. Whole Exome Sequencing (WES) was performed on indexed paired-end libraries (NEB Next DNA Kit) and Agilent SureSelect Human All Exon 50 Mb kit (Agilent) as previously reported (8 (link)). RNA was isolated from separate LCM-captured cells (10 frozen sections) using Qiagen RNeasy mini-kit and established protocol. mRNA (2–10 ng, RIN>6) was amplified using NuGEN Ovation RNA-seq v2 mRNA amplification/cDNA generation kit, before library preparation using Ovation Ultralow DR Multiplex System. Indexed libraries were sequenced on the Illumina HiSeq platform 101bp paired-end reads at 2, 3, or 4 libraries per lane for MPseq, WES, and RNA-Seq, respectively.
Repli g wga kit
Manufactured by Qiagen
The Repli-g WGA kit is a DNA amplification product designed to generate high-quality, whole-genome amplified (WGA) DNA from a small amount of starting material. The kit utilizes a proprietary, isothermal amplification technology to efficiently amplify DNA without the need for thermal cycling equipment.
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Lab products found in correlation
3 protocols using repli g wga kit
1
Profiling PDAC Tumor and PanIN Tissues
2
Single-Cell DNA Extraction and Amplification
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DNA was purified from individual (or few, ca. 10 cells) cell consortia with the PicoPure DNA extraction kit (Applied Biosystems). 16 S rRNA genes were PCR-amplified using the primers B-27F and 1492 R (Supplementary Table 2 ). PCR reactions were performed for 30 cycles (denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min) preceded by 2 min denaturation at 94 °C, and followed by 7 min extension at 72 °C. 16 S rRNA gene clone libraries were constructed with the Topo TA cloning system (Invitrogen) following the instructions provided by the manufacturer. After plating, positive transformants were screened by PCR amplification using M13R and T7 flanking vector primers. 16 S rRNA amplicons were Sanger-sequenced using the 1492 R primer by Genewiz (Essex, UK). Whole genome amplification (WGA) was carried out on PicoPure-extracted DNA using Multiple Displacement Amplification46 (link) (MDA) with the REPLI-g WGA kit (Qiagen) and Multiple Annealing and Looping-Based Amplification Cycles47 (link) (MALBAC) with the MALBAC Single Cell WGA kit (Yikon Genomics).
3
Isolation and Propagation of Rhizophagus irregularis C3
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Rhizophagus irregularis isolate C3 was originally isolated from a Tänikon, Switzerland as described in [37 (link)]. The fungus was propagated on Agrobacterium rhizogenes-transformed Daucus carota root cultures on M medium [57 (link), 62 ].
Single spore lines were generated by placing a single C3 spore next to a fresh D. carota root culture (initial culture provided by dr. Toby Kiers, University of Amsterdam). Spores were selected from spore clusters from the same source plate, and single spore lines were named after their respective cluster.
Medicago selection lines (MedSel) were made by inoculating Medicago truncatula (Jemalong A17) root cultures with ~ 50 C3 spores. When these cultures produced enough spores, these spores moved to fresh M. truncatula root cultures to start a new round. Three of these subsequent transfers were made. For DNA sequencing, ~ 50 spores were isolated from the M medium and crushed in 2 μL DNA free mQ water. Total genomic DNA was then amplified using the Repli-G WGA kit (Qiagen).
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Rhizophagus irregularis isolate C3 was originally isolated from a Tänikon, Switzerland as described in [37 (link)]. The fungus was propagated on Agrobacterium rhizogenes-transformed Daucus carota root cultures on M medium [57 (link), 62 ].
Single spore lines were generated by placing a single C3 spore next to a fresh D. carota root culture (initial culture provided by dr. Toby Kiers, University of Amsterdam). Spores were selected from spore clusters from the same source plate, and single spore lines were named after their respective cluster.
Medicago selection lines (MedSel) were made by inoculating Medicago truncatula (Jemalong A17) root cultures with ~ 50 C3 spores. When these cultures produced enough spores, these spores moved to fresh M. truncatula root cultures to start a new round. Three of these subsequent transfers were made. For DNA sequencing, ~ 50 spores were isolated from the M medium and crushed in 2 μL DNA free mQ water. Total genomic DNA was then amplified using the Repli-G WGA kit (Qiagen).
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