The plates were put in the hypoxia chamber, 1% O2, RT. We collected the worms after 3 and 6 h exposure to 1% O2 (the 3 and 6 h time points, respectively). After 24 h in 1% O2, we placed the remaining plates in 21% O2. We collected the worms after 30 min and 4 h incubation in 21% O2 (the 24.5 h and 28 h time points, respectively). Worms were collected from plates by washing the plates 3 times with ice-cold phosphate-buffered saline (PBS, pH 7.4 supplemented with 1mM MgSO4). The resulted worm pellet was washed for additional 3 times with PBS. To crosslink mRNA with FLAG-PAB-1 in vivo, worms were treated with 1% paraformaldehyde (PFA) in PBS for 1 hour on ice with occasional agitation.
Growth protocol
To synchronize worms, we collected embryos from gravid hermaphrodites as described previously (Brenner, 1974). The collected embryos were then rotated in M9 buffer for 16 h at 21°C. We placed approximately 4800 synchronized L1 larvae in twelve 3.5 cm NGM plates seeded with 250 µl OP50 and grown them for 3 days until they reached the L4 larval stage (the 0 time point).
Extracted molecule
polyA RNA
Extraction protocol
Worms were washed 3 times with PBS and once with homogenization buffer (HB; 150 mM NaCl, 50 mM phosphate buffer pH 7.2, 1 mM EGTA, 15 mM EDTA, 0.6 mg/ml heparin sodium salt, 10% v/v glycerol, RNAsin, 1 mM DTT, 0.5% v/v Igepal (Sigma, #18896), 1:100 protease inhibitors cocktail (Sigma, #P2714). HB was treated with 1:1000 DEPC, and then RNAse free heparin sodium salt was added). Worms were resuspended in HB and homogenized using Bullet Blender (Next Advance) according to manufacturer's protocol. Worm debris was removed by centrifugation at 17,000xg for 20 min. To equilibrate the anti-FLAG beads, we washed them twice with 1 ml of 0.1M glycine, pH 3.5, following 4 washes with 500 µl of HB. All centrifuges of beads were performed at 100xg, 4°C for 3 min. We incubated amount of lysate equivalent to 50 ng of protein with 50 µl of equilibrated beads for 2 h at 4°C, then centrifuged and removed the unbound material. Beads were washed three times with 500 µl of low-salt HB buffer (LSHB; 25 mM NaCl, 20 mM phosphate buffer pH 7.2, 1 mM EGTA, 1 mM EDTA, 0.6 mg/ml heparin sodium salt, 10% v/v glycerol, RNAsin, 1mM DTT, 0.5% v/v Igepal, 1:100 protease inhibitors cocktail (Sigma, #P2714). LSHB was DEPC treated like HB). Beads were incubated with 500 µl of LSHB for 30 min. at 4°C with agitation. To elute precipitated mRNA, beads were incubated with 125 µl Trizol at 65°C for 30 min. with rotation. Elution was repeated with 15-min. incubation. Resulted RNA was purified with RNeasy minelute cleanup kit (Qiagen, #74204) following manufacturer's protocol with one change: to elute RNA, column was incubated for 1 minute with 10mM Tris pH 7.0 preheated to 65°C. The quality of precipitated mRNA was estimated using Bioanalyzer (Agilent).
Label
Cy3
Label protocol
For each sample 10 ng of purified mRNA were amplified and labeled with fluorescent dyes (Cy3 or Cy5) using the Low RNA Input Linear Amplification & Labeling kit (Agilent).
The plates were put in the hypoxia chamber, 1% O2, RT. We collected the worms after 3 and 6 h exposure to 1% O2 (the 3 and 6 h time points, respectively). After 24 h in 1% O2, we placed the remaining plates in 21% O2. We collected the worms after 30 min and 4 h incubation in 21% O2 (the 24.5 h and 28 h time points, respectively). Worms were collected from plates by washing the plates 3 times with ice-cold phosphate-buffered saline (PBS, pH 7.4 supplemented with 1mM MgSO4). The resulted worm pellet was washed for additional 3 times with PBS. To crosslink mRNA with FLAG-PAB-1 in vivo, worms were treated with 1% paraformaldehyde (PFA) in PBS for 1 hour on ice with occasional agitation.
Growth protocol
To synchronize worms, we collected embryos from gravid hermaphrodites as described previously (Brenner, 1974). The collected embryos were then rotated in M9 buffer for 16 h at 21°C. We placed approximately 4800 synchronized L1 larvae in twelve 3.5 cm NGM plates seeded with 250 µl OP50 and grown them for 3 days until they reached the L4 larval stage (the 0 time point).
Extracted molecule
polyA RNA
Extraction protocol
Worms were washed 3 times with PBS and once with homogenization buffer (HB; 150 mM NaCl, 50 mM phosphate buffer pH 7.2, 1 mM EGTA, 15 mM EDTA, 0.6 mg/ml heparin sodium salt, 10% v/v glycerol, RNAsin, 1 mM DTT, 0.5% v/v Igepal (Sigma, #18896), 1:100 protease inhibitors cocktail (Sigma, #P2714). HB was treated with 1:1000 DEPC, and then RNAse free heparin sodium salt was added). Worms were resuspended in HB and homogenized using Bullet Blender (Next Advance) according to manufacturer's protocol. Worm debris was removed by centrifugation at 17,000xg for 20 min. To equilibrate the anti-FLAG beads, we washed them twice with 1 ml of 0.1M glycine, pH 3.5, following 4 washes with 500 µl of HB. All centrifuges of beads were performed at 100xg, 4°C for 3 min. We incubated amount of lysate equivalent to 50 ng of protein with 50 µl of equilibrated beads for 2 h at 4°C, then centrifuged and removed the unbound material. Beads were washed three times with 500 µl of low-salt HB buffer (LSHB; 25 mM NaCl, 20 mM phosphate buffer pH 7.2, 1 mM EGTA, 1 mM EDTA, 0.6 mg/ml heparin sodium salt, 10% v/v glycerol, RNAsin, 1mM DTT, 0.5% v/v Igepal, 1:100 protease inhibitors cocktail (Sigma, #P2714). LSHB was DEPC treated like HB). Beads were incubated with 500 µl of LSHB for 30 min. at 4°C with agitation. To elute precipitated mRNA, beads were incubated with 125 µl Trizol at 65°C for 30 min. with rotation. Elution was repeated with 15-min. incubation. Resulted RNA was purified with RNeasy minelute cleanup kit (Qiagen, #74204) following manufacturer's protocol with one change: to elute RNA, column was incubated for 1 minute with 10mM Tris pH 7.0 preheated to 65°C. The quality of precipitated mRNA was estimated using Bioanalyzer (Agilent).
Label
Cy5
Label protocol
For each sample 10 ng of purified mRNA were amplified and labeled with fluorescent dyes (Cy3 or Cy5) using the Low RNA Input Linear Amplification & Labeling kit (Agilent).
Hybridization protocol
Labeled cRNA was hybridized to the Agilent C. elegans (V2) Gene Expression Microarray, 4x44K (design 020186, Agilent Technologies, USA) for 17 hours at 65°c in an Agilent DNA-Microarray Hybridization Oven. The arrays were later washed using the Gene Expression Wash Buffer Kit.
Scan protocol
The Microarray was scanned using the Agilent High-Resolution Microarray Scanner and the data was extracted from the resulting images using the Feature Extraction 10.7 software.
Description
a fluorescence read above 5000 intensity units in at least one npr1 sample
Data processing
The resulting txt files have been analyzed utilizing custom written programs in MATLAB R2013A, using routines from the Bioinformatics and Statistical toolbox. In every individual array from a specific time point the columns of median Cy3 and Cy5 were selected and the corresponding median background of Cy3 and Cy5 was subtracted. Averaging was carried out on the 45,220 probes of the array (using the consolidation function of Microsoft Excel). This resulted in 21,268 genes in every array. Lowess normalization was carried out and flooring was performed on each of the 2 columns, set to 20 measurements. To enrich for abundant transcripts and robust changes in expression levels, data was filtered by raw data read values, requiring minimal value in at least one sample as described in the description column.
