2 protocols
normalization data transformation protocol
Base calls were generated using Illumina’s Casava 1.8.2 pipeline Paired-end reads were aligned to mm9 using tophat 2.0. Non-default parameters include –mate-inner-dist -40 –mate-std.dev 50 –read-mismatches 5 – read-edit-dist 5 RefSeq genes were quantified using the summarizeOverlaps fucntion within the GenomeRanges Bioconductor package. Differential genes were identified using edgeR from Biocoductor fdr 0.0005 Genome_build: mm9 Supplementary_files_format_and_content: 'raw.gene.counts.txt': non-normalized fragment counts per gene, which was used as input into edgeR for differential analysis 'normalized.pergene.counts.txt': normalized fragment counts per-gene, log2 fold changes and p-values
nucleic acid library construction protocol
1. Homogenization: Cells grown in monolayer were lysed directly in the culture dish by the addition of RNA-Bee with 1 ml of the reagent for a 6-well plate well. Pass the lysate through a pipette several times to ensure lysis. 2. Phase Separation: Add 0.2 ml chloroform per 1 ml of RNA-Bee, cap the tube and vortex for 15-30 seconds. Store the sample on ice for 5 minutes. Centrifuge the homogenate at 12,000g for 15 minutes at 4 degrees C. Following centrifugation, the sample forms the lower blue phenol-chloroform phase, interphase, and the upper colorless aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 50% of the initial volume of RNA-Bee plus sample volume. Chloroform should not contain isoamyl alcohol or any other additives. 3. RNA Precipitation: Transfer the aqueous phase to a clean tube, add 0.5 ml of isopropanol, and store the sample for 5-10 minutes at room temperature. Centrifuge at 12,000g for 5 minutes at 4 degrees C. RNA precipitate (often not visible before centrifugation) forms a white-yellow pellet at the bottom of the tube. 4. RNA Wash: Remove the supernatant and washs the RNA pellet with 70% ethanol, shaking or vortexing to dislodge the pellet from the side of the tube. Centrifuge for 5 minutes at 12000g at 4 degrees C. Use at least 1 ml of ethanol solution per 1 ml of RNA-Bee used for the initial homogenisation. 5. RNA Solubilization: At the end of the procedure, briefly air-dry the RNA pellet (5-10 minutes). It is important not to let the RNA pellet dry completely, as this greatly decreases its solubility. Dissolve the RNA in water and incubate for 10-15 minutes at 55 degrees C. TruSeq Stranded mRNA protocol from Illumina