Dna enzymatic labelling kit

Worms were collected as young adults 46 h after a 1 h synchronization by collecting hatching L1 larvae as described above. Single worms were then washed, collected in a 1.5 ml microcentrifuge tube (Eppendorf) and snap-frozen in liquid nitrogen. Total RNA was extracted using an RNAClean XP kit (Agentcourt) following the manufacturer’s instructions and amplified using the TransPlex Complete Whole Transcriptome Amplification Kit (Sigma Aldrich) to generate cDNA. Cy3-labeled cDNA was prepared from 500 ng of double-stranded cDNA using the DNA Enzymatic Labelling Kit (Agilent) according to the manufacturer’s instructions, followed by purification with a 30 kDa column (Amicon). Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer (ThermoScientific). cDNA was mixed with hybridization buffer and blocking agent (Agilent) and incubated at 95 °C for 3 min before cooling on ice. cDNA was then hybridized to a custom C. elegans 4x44K microarray (Agilent) for 40 h at 65 °C. Microarrays were washed 1 min at room temperature with GE wash buffer 1 (Agilent) and 1 min at 37 °C with GE wash buffer 2 (Agilent), then dried immediately by brief centrifugation. Microarrays were scanned on a G2539A scanner (Agilent) at 5 µm resolution and 100 % PMT. Probe intensities were extracted and their quality was assessed with the Feature Extraction software 10.7 (Agilent).