strain: Roughwood 40-11 elapsed time (hours): 28 day: Day02 zeitgeber time: ZT20 light cycle phase: dark period
Treatment protocol
Third generation females (7–9 day old) were maintained at 18˚C in 750mL of COMBO medium with a 12L:12D photoperiod (with abrupt transitions). Replicates of 30 individuals were collected and immediately frozen every 4 hours over a 48 hour time course. Period measurements are labeled as hour of collection and phase of the light cycle.
Growth protocol
Daphnia cultures of genotype, Roughwood 40-11, were maintained for three generations at 18°C in 750mL of COMBO medium (Kilham, Kreeger, Lynn, Goulden, & Herrera, 1998) with a 12L:12D photoperiod (with abrupt transitions). Every second day, culture media containing Scenedesmus acutus algae (200,000 cells/ml) was replaced, and individuals were culled to maintain a density of 50 individuals per 750mL.
Extracted molecule
total RNA
Extraction protocol
Samples were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA). The homogenate was purified using Qiagen’s RNeasy Mini Kit (Qiagen,Venlo, Netherlands) with on-column DNAse treatment to isolate total RNA (Lopez & Colbourne 2011).
Label
CY5
Label protocol
Beginning with 1.0 μg of total RNA, a single round of amplification using MessageAmpTM II aRNA kit (Ambion, Austin, TX) was performed for each RNA sample. The cRNA (10 μg) was converted to double strand cDNA with random primers using the Invitrogen SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA). From 1 μg double-stranded cDNA, labeled cDNA was generated with NimbleGen’s Dual-colour Labeling Kit (Roche NimbleGen, Inc.).
strain: Roughwood 40-11 elapsed time (hours): 24 day: Day02 zeitgeber time: ZT16 light cycle phase: dark period
Treatment protocol
Third generation females (7–9 day old) were maintained at 18˚C in 750mL of COMBO medium with a 12L:12D photoperiod (with abrupt transitions). Replicates of 30 individuals were collected and immediately frozen every 4 hours over a 48 hour time course. Period measurements are labeled as hour of collection and phase of the light cycle.
Growth protocol
Daphnia cultures of genotype, Roughwood 40-11, were maintained for three generations at 18°C in 750mL of COMBO medium (Kilham, Kreeger, Lynn, Goulden, & Herrera, 1998) with a 12L:12D photoperiod (with abrupt transitions). Every second day, culture media containing Scenedesmus acutus algae (200,000 cells/ml) was replaced, and individuals were culled to maintain a density of 50 individuals per 750mL.
Extracted molecule
total RNA
Extraction protocol
Samples were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA). The homogenate was purified using Qiagen’s RNeasy Mini Kit (Qiagen,Venlo, Netherlands) with on-column DNAse treatment to isolate total RNA (Lopez & Colbourne 2011).
Label
CY3
Label protocol
Beginning with 1.0 μg of total RNA, a single round of amplification using MessageAmpTM II aRNA kit (Ambion, Austin, TX) was performed for each RNA sample. The cRNA (10 μg) was converted to double strand cDNA with random primers using the Invitrogen SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA). From 1 μg double-stranded cDNA, labeled cDNA was generated with NimbleGen’s Dual-colour Labeling Kit (Roche NimbleGen, Inc.).
Hybridization protocol
Dual-colour hybridization, post-hybridization washing, and scanning were done according to manufacturer's instructions (Roche NimbleGen, Madison, WI).
Scan protocol
Images were acquired using a NimbleGen MS 200 with 2μm resolution (Roche NimbleGen, Madison, WI).
Data processing
The image data from these arrays were processed using the software NimbleScan v2.5 (Roche NimbleGen, Madison, WI).
