A 3'-end-biotinylated miR-155 (10 nM, M5-M6, M9) or biotinylated cel-miR-67 control (M7-M8) was incubated with S9 lung epithelial cells for 24 hours, and cell lysates were enriched for mRNA:miR-155 and mRNA:cel-miR67 complexes, respectively, using streptavidin bead-based affinity purification. S9 cells were treated with with miR-155-mimic (10 nM, M1-M2) or control mimic (M3-M4) for 48 hours
Growth protocol
CFTR-repaired IB3-1/S9 cells were maintained in LHC-8 serum-free medium (Invitrogen, 12678-017) in humidified 5% CO2. IB3-1/S9 cells (3.0x 10^6) were plated on one 100-mm dish and incubated in LHC-8 media for 24 hours, after which cells were transfected with 10 nM of 3'-biotinylated miR-155 (Dharmacon) or biotinylated cel-miR-67 control using siPORT-NeoFX (Invitrogen, AM4511). After a 24 hour incubation, cells were washed twice with PBS and lysed with 700 µl lysis buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100mM KCl, 0.3% NP40), including 50U RNaseOUT (Invitrogen, 10777-019) and proteinase inhibitor (Sigma-Aldrich, S8820). Cells were collected by scraping the dish, and the collected cells were incubated on ice for 5 minutes before centrifugation at 10000×g for 5 min at 4°C, and collection of the supernatant for streptavidin purification. Streptavidin-coated magnetic beads (Invitrogen, 11205D) were incubated for 2 hours at 4°C with blocking buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.3% NP40, 1 mg/ml yeast tRNA, 1 mg/ml BSA). The cell lysate was then added to the magnetic beads, and incubated for 4 hours at 4°C, after which the beads were rinsed five times with lysis buffer, and RNA isolated from the beads. For the second set of experiments, IB3-1/S9 cells (2.5x105) were plated on a 6-well plate in LHC-8 media for 24 hours, after which 10 nM miR-155 mimic or control mimic was transfected into cells with siPORT-NeoFX. RNA was isolated from these cells 48 hours after transfection.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the miRVana kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's instructions.
Label
Cy3
Label protocol
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5 μg of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
Standard Illumina protocol. In short, a total of 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's Sentrix Mouse Red-8 v3 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.53 μm using the Illumina iScan scanner.
Description
S9 + Bi MiR-155 IP-M9
Data processing
Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores is supplied.The resulting dataset was analyzed with DIANE 6.0, a spreadsheet-based microarray analysis program. Using DIANE, the results were normalized by Z-Score transformation. Z-normalized data were then analyzed with principal component analysis (PCA). To determine the gene expression changes within each specific RNA comparison, Z-Scores for paired treatment groups were compared using the Z-Ratio statistic. Expression changes for individual genes were considered significant if they met four criteria: Z-Ratio above 1.5 or below -1.5; false detection rate (FDR) of less than 0.30; a P-value statistic for Z-Score replicability below 0.05; and mean background-corrected signal intensity greater than zero. Differentially expressed genes were identified as significant (p < 0.05) based on Z-scores. Gene set analysis was performed using GO gene sets with the PAGE algorithm. All Venn diagrams of gene expression were generated using the online tool Venny.
