4 protocols
Sequences were aligned to the July 2007 assembly of the mouse genome (NCBI37/mm9) using GSNAP (Wu and Nacu, Bioinformatics 26:873-81), where annotated splice junctions were provided from Ensembl Build 63 (Flicek et al., Nucleic Acids Res. 40:D84-90) and up to five mismatches were allowed. Transcript quantification was performed using htseq-count, part of the HTSeq package for the R statistical computing platform. Differentially expressed genes were identified with the Bioconductor package DESeq (Anders and Huber, Genome Biol. 11:R106), and Gene Ontology (GO) enrichment analysis was performed using GOstats (Falcon and Gentleman, Bioinformatics 23:257-8). The reported P-values were corrected for multiple testing using the Benjamini & Hochberg method.
Sequencing was performed on the Illumina GAIIx yielding 38-41M single-end 105bp reads per library.
Total RNA was extracted using the TRIzol method followed by treatment with TURBO DNase (Ambion). Polyadenylated transcripts were selected from 2 ug total RNA using Sera-Mag beads (Thermo Scientific). Between 60 and 100 ng mRNA was then sheared to approximately 200 nt fragments by focused ultrasound on the Covaris S2 using the following parameters: Duty Cycle =10%, Intensity = 5, Cycles Per Burst = 200, for 75s with frequency sweeping enabled. First-strand cDNA synthesis was performed at 50 degrees C for 2 hours using SuperScript III (Invitrogen) and random hexamer primers, followed by second-strand synthesis with DNA Polymerase I at 16 degrees C for 2 hours in the presence of RNaseH. End repair of double-stranded cDNA products was carried out with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs). Blunted, phosphorylated cDNA fragments were then 3'-adenylated via Klenow fragment and ligated to sequencing adapters (Illumina) by T4 DNA ligase at 20 degrees C for 30 minutes. PCR amplification of library constructs was carried out with Phusion DNA polymerase (Finnzymes) for 13 cycles. Purification of reaction products between each step was performed with Ampure XP paramagnetic beads (Beckman Coulter). Prior to sequencing, the molarity and size distribution of the libraries was assessed by DNA 1000 microfluidic chips on the Agilent 2100 Bioanalyzer.
ES cells were cultured in 2i defined medium, a 1:1 mixture of DMEM/F-12 and Neurobasal with N2 and B27 supplements, 1uM PD0325901 and 3uM CHIRON99021.