Northern blotting was performed as previously see more
described (Gerber et?al., 2001) by separating total RNA on a formaldehyde-containing 2% agarose gel and transferring to Hybond N membrane (GE Healthcare). Northern blots were hybridized at 65��C in 50% formamide-containing hybridization buffer, with 32P-labeled riboprobes complementary to U11 (nt 24�C156) and U6 (nt 1�C82) and a probe to Rp49 (Gerber et?al., 2001). Northern blots were probed sequentially with U11, Rp49, and U6 probes. Probe signal was imaged on a phosphor storage screen from 3 to 90?min, scanned on a Typhoon Trio (GE Healthcare), and quantitated with ImageQuant software at exposures without saturated pixels. Affymetrix Drosophila Genome 2 arrays were analyzed in R, version 2.11.1, using the packages affy ( Gautier et?al., 2004), version 1.26.1, and limma ( Smyth et?al., 2005), version 3.4.3. Normalization was done using rma. Annotation information for the probes was taken from Ensembl 62. RNAi was performed with the ELL-A dsRNA. A region of the B-lactamase gene was used as a nontargeting control. Two micrograms of DNase-treated total RNA was depleted of Ribosomal RNA with the Ribo-Zero kit from Epicenter, and library preparation was made using the Tru-seq mRNA kit from Illumina. Sequencing reads were acquired through the primary Solexa image analysis pipeline, where bases were called and reads were filtered for quality, according to default Solexa standards. Filtered reads were then aligned to the fly genome Bumetanide
(UCSC dm3) or mouse genome (UCSC mm9) using the Bowtie (Langmead et?al., 2009) alignment tool, version 0.12.7. Only those sequences that matched uniquely to the genome with up to two mismatches were retained for subsequent analyses. Enriched regions of ChIP-seq signal were determined by the ��MACS�� (Zhang et?al., 2008) peak-finding program, version 1.4.0rc2. Sequence reads for each ChIP-seq data set and its associated whole-cell extract controls were used for the input and control file, Alisertib concentration
respectively. The effective genome size was configured appropriately for the fly data sets, and the p value cutoff was set to 1.00e-05 and a fold change greater than five, or FDR?< 1%. All other MACS parameters were left default. RNA-seq analysis was done using TopHat v1.2.0 (Trapnell et?al., 2009) and Bowtie v0.12.7. Only uniquely mapping reads were used. Fly transcript annotations were from Ensembl 62, and mouse transcript annotations were from Ensembl 63. Differentially expressed genes after RNAi were called with an adjusted p value?< 0.001, and MA plots were determined by DESeq (Anders and Huber, 2010), v1.4.1. For the Drosophila RNA-seq experiments, two different dsRNA regions for each target were treated as biological replicates. A nontargeting control dsRNA-treated sample and untreated S2 cells were treated as biological replicates for the ��control�� condition.