INS-1 cells were induced with 1ug/mL Doxycycline for 24 hours, then either left untreated for UT samples, or treated with 1uM Thapsigargin for 30 minutes.
Growth protocol
INS-1 cells with doxycycline inducible expression of EGFP or EGFP-L10a fusion generated from INS-1/FRT/TO cells (#5-3.19) (Thomas et al., 2004) were grown in RPMI, 10% fetal calf serum, 1mM sodium pyruvate, 10mM HEPES, 2mM glutamine, 50µM b-mercaptoethanol, 10µg/ml blasticidin.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol. For Ribosomal immunoprecipiation, after treatment with Tg, cells were then treated with 100 μg/ml cycloheximide for 20 minutes, then washed twice with PBS containing 100 μg/ml cycloheximide. Lysis buffer was added (20mM HEPES [pH 7.4], 150mM KCL, 2M MgCl2, 1% NP-40—a non-ionic, non-denaturing detergent purchased from EMD Biosciences, San Diego, CA), and plates were incubated on ice for 10 minutes before detaching cells with cell lifters. Lysates were immediately homogenized in ice-cold polysome extraction buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, protease inhibitors, and recombinant RNase inhibitors) using a motor-driven Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet large cell debris, and NP-40 and 1,2-Diheptanoyl-sn-Glycero-3-Phosphocholine (DHPC) (Avanti Polar Lipids, Alabaster, AL) were added to the supernatant at a final concentration of 1% and 30 mM, respectively, to extract polysomes. After incubation on ice for 5 minutes, the clarified lysate was centrifuged for 10 minutes at 13,000 ×g to pellet insoluble material. Goat anti-GFP (custom made in laboratory of Nathaniel Heintz, Rockefeller Univ.) coated protein G Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) were added to the supernatant and the mixture was incubated at 4°C with end-over-end rotation for 30 minutes. Beads were subsequently collected on a magnetic rack, washed three times with high-salt polysome wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM dithiothreitol, 100 μg/ml cycloheximide) and immediately placed in TriZol-LS reagent (Invitrogen Corporation, Carlsbad CA) and chloroform to extract the bound rRNA and mRNA from polysomes. After extraction, RNA was precipitated with sodium acetate and Glycoblue (Ambion, Austin, TX) in isopropanol overnight at −80°C, washed twice with 70% ethanol, resuspended in water, followed by a DNase digestion, and ethanol purification, before being resuspended in water, and quantitated using a Nanodrop.
Label
Cy3
Label protocol
After cDNA purification, cDNA was labeled with either Cy3 (reference) or Cy5 (treated sample) using the Amersham Cy dye kit. 1 Dye tube was dissolved in 30uL DMSO, and 10uL Cy Dye was mixed with 10uL cDNA from sample. Samples were incubated for 1 hour in the dark, and then reactions were purified using the Qiagen MiniElute DNA purification columns, and eluted in 12uL H20. The untreated Cy3 sample was then mixed with the respective treated sample (Cy5), and incubated on ice until hybridization.
INS-1 cells were induced with 1ug/mL Doxycycline for 24 hours, then either left untreated for UT samples, or treated with 1uM Thapsigargin for 30 minutes.
Growth protocol
INS-1 cells with doxycycline inducible expression of EGFP or EGFP-L10a fusion generated from INS-1/FRT/TO cells (#5-3.19) (Thomas et al., 2004) were grown in RPMI, 10% fetal calf serum, 1mM sodium pyruvate, 10mM HEPES, 2mM glutamine, 50µM b-mercaptoethanol, 10µg/ml blasticidin.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol. For Ribosomal immunoprecipiation, after treatment with Tg, cells were then treated with 100 μg/ml cycloheximide for 20 minutes, then washed twice with PBS containing 100 μg/ml cycloheximide. Lysis buffer was added (20mM HEPES [pH 7.4], 150mM KCL, 2M MgCl2, 1% NP-40—a non-ionic, non-denaturing detergent purchased from EMD Biosciences, San Diego, CA), and plates were incubated on ice for 10 minutes before detaching cells with cell lifters. Lysates were immediately homogenized in ice-cold polysome extraction buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 μg/ml cycloheximide, protease inhibitors, and recombinant RNase inhibitors) using a motor-driven Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 × g, 4 °C, to pellet large cell debris, and NP-40 and 1,2-Diheptanoyl-sn-Glycero-3-Phosphocholine (DHPC) (Avanti Polar Lipids, Alabaster, AL) were added to the supernatant at a final concentration of 1% and 30 mM, respectively, to extract polysomes. After incubation on ice for 5 minutes, the clarified lysate was centrifuged for 10 minutes at 13,000 ×g to pellet insoluble material. Goat anti-GFP (custom made in laboratory of Nathaniel Heintz, Rockefeller Univ.) coated protein G Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) were added to the supernatant and the mixture was incubated at 4°C with end-over-end rotation for 30 minutes. Beads were subsequently collected on a magnetic rack, washed three times with high-salt polysome wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM dithiothreitol, 100 μg/ml cycloheximide) and immediately placed in TriZol-LS reagent (Invitrogen Corporation, Carlsbad CA) and chloroform to extract the bound rRNA and mRNA from polysomes. After extraction, RNA was precipitated with sodium acetate and Glycoblue (Ambion, Austin, TX) in isopropanol overnight at −80°C, washed twice with 70% ethanol, resuspended in water, followed by a DNase digestion, and ethanol purification, before being resuspended in water, and quantitated using a Nanodrop.
Label
Cy5
Label protocol
After cDNA purification, cDNA was labeled with either Cy3 (reference) or Cy5 (treated sample) using the Amersham Cy dye kit. 1 Dye tube was dissolved in 30uL DMSO, and 10uL Cy Dye was mixed with 10uL cDNA from sample. Samples were incubated for 1 hour in the dark, and then reactions were purified using the Qiagen MiniElute DNA purification columns, and eluted in 12uL H20. The untreated Cy3 sample was then mixed with the respective treated sample (Cy5), and incubated on ice until hybridization.
Hybridization protocol
CyDye conjugated sample (24uL) was added to 30uL 2x hybridization buffer (50% formamide, 10X SSC, 0.2% SDS, H20), OligoDT(3uL), and COtI DNA(3uL-Invitrogen), and then probe was denatured at 95 degrees Celsius for 5 minutes. Tubes were quickly spun down and samples were kept at 42C until added to Pancchip 6.1 using the MAUI hybrization system (Biomicro systems) overnight at 42C. .
Scan protocol
Slides were washed and then scanned using an Axon scanner and data analyzed using the GenePixPro software.
Data processing
We normalized the data using the NOMAD database, and filtered to remove spots with low signal intensity (<2 fold above background) or inconsistent Cy5 to Cy3 signal ratios within the spot (R2 >0.7 for linear relationship between Cy5 vs. Cy3 signal). We then calculated the log2 ratio of the induced to uninduced signals (normalized to the Cy3 reference sample) for each target gene, and averaged the ratios from three independent experiments for each condition. We used Gene Cluster 3.0 to perform hierarchical clustering of genes induced and downregulated at least two-fold and used the Treeview software to visualize the clustered data.
