Advantage 2 pcr kit

Total RNA was converted to cDNA using SMARTer PCR cDNA synthesis kit and amplified with the Advantage 2 PCR kit (Clontech) and then purified with a QIAquick PCR Purification Kit (QIAGEN). cDNA quantity was determined with a Nanodrop 8000 (Thermo Scientific) and size distribution with the Agilent High Sensitivity DNA kit (Agilent Technologies). Gene expression was measured using the Gene Expression Master Mix (Applied Biosystems) and predesigned TaqMan real-time PCR assays for the following targets: AHR (Hs00169233_m1), HSP90AB1 (Hs00607336_gH), ACVR2A (Hs01012007_m1), IVNS1ABP (Hs01573482_m1), and HPRT1 (Hs02800695_m1) on ABI PRISM 7900HT Sequence Detection System (Thermo Scientific). Reactions were set up in duplicate in a 384-well plate with 40 ng cDNA per well in 20 µl volume, and the data were analyzed using RQ Manager 1.2.1 (Thermo Scientific). All experimental procedures were conducted according to the manufacturer’s instructions.

For N. brasiliensis, three small (5–10 μm) C1 Single-Cell Auto Prep IFC chips (Fluidigm) were primed and 5000 cells were sorted directly into the chip. For P. chabaudi, cells were loaded at a concentration of 1700 cells μl^-1 onto C1 small chips. To allow estimation of technical variability, 1 μl of a 1:4000 dilution of ERCC (External RNA Controls Consortium) spike-in mix (Ambion, Life Technologies) was added to the lysis reagent. Cell capture sites were visually inspected one by one using a microscope. The capture efficiency is described in Additional file 2: Tables S1 and S7.
The capture sites that did not contain single cells were noted and were removed from downstream analysis. Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech) and the Advantage 2 PCR kit according to the manufacturer’s instructions on the C1 device. cDNA was harvested and diluted to 0.1–0.3 ng/μl and libraries were prepared in 96-well plates using a Nextera XT DNA Sample Preparation kit (Illumina) according to the protocol supplied by Fluidigm. Libraries were pooled and sequenced on an Illumina HiSeq2500 using paired-end 75-bp reads for N. brasiliensis and 100-bp reads for P. chabaudi.

To assess SMART-ddPCR measurements of AI using an alternative method, we carried out PCR and Sanger sequencing for one deletion locus, IKZF1 SNP rs4132601, and one copy number gain locus, ARID5B SNP rs7089424. PCR primers were designed using the Primer3 software (http://primer3.ut.ee) and PCR reactions carried out using the Advantage 2 PCR kit (Clontech) for 6 constitutional and 13 tumor DNA samples heterozygous for rs4132601, and for 7 constitutional and 17 tumor samples heterozygous for rs7089424. PCR products were cleaned up using ExoSAP-IT reagent (Affymetrix), and sequenced bi-directionally using an ABI 3730xl DNA sequencer. Sequence chromatogram files were analyzed using Chromas software (Technelysium). For each sample, SNP risk allele proportions were calculated for both forward and reverse sequences as follows:
$Riskalleleproportion=\frac{PeakheightofSNPriskallele}{(PeakheightofSNPriskallele+peakheightofSNPprotectiveallele)}$
The mean risk allele proportion across forward and reverse sequences was then determined. As described above for SMART-ddPCR, thresholds of AI were calculated from repeat measurements on constitutional DNA from a subset of heterozygote cases.

Total RNA was extracted from RNAlater®-preserved samples using the Ambion RNAqueous®-Micro Total RNA Isolation kit. First-strand cDNA was constructed using the SMART® cDNA Library Construction Kit (Clontech Laboratories, Inc.), replacing the included 3′ primer with the Cap-TRSA-CV oligo [19 (link)]. We amplified double-stranded cDNA using the Advantage® 2 PCR Kit (Clontech Laboratories, Inc.). To minimize the risk of contamination, extractions and cDNA construction were performed in small batches of four tissue samples or fewer, and the workstation and tools were cleaned with bleach between each set of extractions. Where possible, we avoided sampling the external body surface and the gut to limit the potential for contamination from epibionts and gut contents (e.g., prey items and microorganisms).
Non-normalized cDNA libraries were sent to Hudson Alpha Institute for Biotechnology, Huntsville, Alabama USA for library preparation and 2 × 100–bp paired-end sequencing on an Illumina HiSeq 2000. Approximately one-sixth of a lane was used for each taxon.

According to the Iso-Seq protocol, 1 µg total RNA was transcribed to generate full-length cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, CA, USA). Then, the cDNA was amplified using the advantage 2 PCR kit (Clontech, CA, USA), and PCR products were purified with AMpure PB beads (Beckman Coulter, CA, UAS). Purification was followed by size selection using the BluePippinTM Size Selection System (Sage Science, MA, USA) of the following bins: 1-2, 2-3 and 3-6 kb. The three libraries were then constructed using SMRTbell Template Prep kit (Pacific Biosciences, CA, USA). Before sequencing, the quality of the libraries was assessed by Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and Qubit fuorometer 2.0 (Life Technologies, CA, USA). Libraries were prepared for sequencing by annealing a sequencing primer and adding polymerase to the primer annealed template. The polymerase-bound template was bound to MagBeads and a total of 6 SMRT cells were sequencing on PacBio RS II platform using P6-C4 chemistry (2 cell each library).

Single E13.5 PGCs were captured on a medium-sized (10–17-μm cell diameter) integrated fluidic circuit (IFC, Fluidigm). Cells were loaded onto the IFC chip at a concentration of 1000 cells/μl, simultaneously stained for cell viability using a LIVE/DEAD Cell Viability/Cytotoxicity Kit (Invitrogen), and observed by fluorescence microscopy to assess the number and viability of cells per capture site. For single PGC RNA-seq, cDNA synthesis and pre-amplification were performed on an IFC chip using the C1 Single-Cell Auto Prep System method (Fludigm) with a SMARTer Ultra Low Input RNA Kit and Advantage 2 PCR Kit (Clontech USA), respectively.