C. elegans was exposed to a sodium hypochlorite-sodium hydroxide solution to obtain viable eggs. The eggs were incubated overnight in M9 buffer at 25°C for hatching, and the suspension of L1-stage worms was centrifuged at 1,200 × g for 2 min. After eliminating the supernatant, the remaining larvae were transferred to fresh mNGM plates seeded with E. coli OP50 and incubated at 25°C. The worms were allowed to grow on E. coli OP50 until the L4 stage. All experiments used 3-day-old worms (1 day adult) to regulate the reproductive system in C. elegans. About 600 worms were transferred to the plates containing 0, and 200 μM of soraprazan, and seeded with E. coli OP50. The plates were incubated at 25°C during 14 days.
Growth protocol
C. elegans Bristol strain N2 provided by the CGC was used. Worms were maintained and propagated in peptone-free mNGM (modified nematode growth medium) at 25°C using standard techniques. E. coli OP50 suspended in M9 buffer was seeded on mNGM in 90 mm diameter petri dishes to feed the worms. Escherichia coli OP50 was provided by the Caenorhabditis Genetics Centre (CGC) at the University of Minnesota and used as a standard food for C. elegans. E. coli OP50 was grown in Luria-Bertani (LB) broth (Difco, Detroit, MI, USA) at 37°C with shaking overnight, collected by centrifugation, and washed twice with M9 buffer. Then, the bacteria were diluted to a final concentration of 0.1 mg (wet weight) per mL in M9 buffer.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
Cy3
Label protocol
Amplified and labeled cRNA was purified on cRNA Cleanup Module (Agilent Technology) according to the manufacturer’s protocol. Labeled cRNA target was quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
Hybridization protocol
After checking labeling efficiency, fragmentation of cRNA was performed by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60oC for 30 min. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto assembled Agilent’s C. elegans Oligo Microarray (44K). The arrays hybridized at 65oC for 17 hours using Agilent Hybridization oven (Agilent Technology, USA). The hybridized microarrays were washed as the manufacturer’s washing protocol (Agilent Technology, USA).
Scan protocol
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA).
Description
Gene expression after 14 days grown on 200 μM soraprazan plates
Data processing
Data was extracted using GeneSpringGX 7.3 (Agilent Technology, USA). Raw intensity data was globally normalized (Cheadle et al.(2003).
