Nimblegen one color dna labeling kit

To select probes for the construction of a secondary array, CS, M808, and 14 randomly selected RILs were genotyped. After digestion of total DNA with PstI and BstNI, adapter ligation, PCR amplification and purification of PCR products as described in the library preparation section, they were labelled with Cy3 using the NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the manufacturer's instructions. Hybridization was performed at 42°C for 72 h on a NimbleGen Hybridization System (Roche Diagnostics). Arrays were scanned using a NimbleGen MS200 Microarray Scanner (Roche Diagnostics) and genotype calling was performed based on the signal intensities.
For selection of probes, a χ² test was used to compute segregation of each probe in the 14 RILs. Those deviating from Mendelian 1 : 1 segregation at a 1% significance level were discarded. The secondary array was constructed using one selected probe per contig or read, consisting of 21,346 CS-derived probes, 21,205 M808-derived probes, and 2,303 probes for normalization. These 44,854 probes were spotted in triplicate on a NimbleGen 12 × 135 K array (Roche Diagnostics). This array was used for genotyping of 210 RILs and the two parental lines.

A custom microarray was made using the fabrication and design services of Roche NimbleGen [103 ]. The array contained gene models for 50,690 contigs and controls, each of which was represented by 7 individual 60-mer homologous probes. For hybridizations, total RNA was extracted from young leaf tissues of plants at 3, 2, 1.5, 1, 0.75, and 0.25 g H₂O/g tissue, as well as of plants rehydrated for 12 or 24 h. RNAs were sent to Roche NimbleGen, where cDNA synthesis, biotin-labeling, and hybridization were performed according to the manufacturer’s protocols. Briefly, total RNA (15 μg) from three biological replicates, each replicate consisting of pooled tissue from three individual plants, of fully hydrated (96% RWC), dehydrating 80%, 60%, 40, 30% RWC, and desiccated 11% RWC, and rehydrated (12 h and 24h) for a total of 24 samples, were converted to biotin-labeled cRNA using the SuperScript double-stranded cDNA Synthesis Kit (Invitrogen), labeled with a NimbleGen One-Color DNA Labeling Kit, and hybridized to arrays using NimbleGen hybridization platform (Roche NimbleGen). Array scanning was performed using the GenePix 4000B Microarray Scanner and data were collected using Roche NimbleGen software.

Total RNA (10 µg each) was reverse transcribed with oligo-dT primer (100 pmol) and the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Life Technologies Co., Grand Island, NY) for 1 hour at 42°C. After the reverse transcription process, a second strand of cDNA was synthesized from the single strand cDNA by using he SuperScript Double-Stranded cDNA Synthesis Kit according to the manufacturer’s procedure (Invitrogen) and after second strand synthesis, total RNA was removed by using RNaseA. Labeled cDNA was synthesized by using a NimbleGen One-Color DNA Labeling Kit (Roche NimbleGen Inc., Madison, WI) including cyanine 3-CTP labeled primer and Klenow enzyme (3′->5′ exo-) according to the manufacturer’s instructions. Labeled cDNA was purified by ethanol precipitation and quantified by the NanoDrop Spectrophotometer (Thermo Scientific Wilmington, DE).

Microarray experiments were conducted by KangChen Bio-technology Co. (Shanghai, China) according to the standard procedure [11 (link)]. Briefly, total RNAs from testes were extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturers’ instructions. Genomic DNA contamination was removed by DNase treatment. The concentration of RNA was measured through a Nanodrop ND-1000 Spectrophotometer (NanoDrop products, Wilmington, DE, USA). Precisely 10 mg of total RNA was taken to synthesize double-stranded cDNA, following the steps of an Invitrogen Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen). RNA contamination was removed by RNase A, and cDNA was precipitated and labelled using a NimbleGen One-Color DNA Labeling Kit (RocheNimbleGen, Inc., Pleasanton, CA, USA). Labelled cDNA was quantitated and hybridized with microarray chip slides in the NimbleGen Hybridization System (Roche-NimbleGen, Inc.). Following hybridization and washing, the microarray slides were scanned with an Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA).