normalization data transformation protocol
Alignment coordinates were compared to the exon coordinates of RefSeq transcripts (Pruitt et al, 2007) and for each transcript the counts of overlapping alignments were recorded. Finally, counts of reads aligning to splice junctions were aggregated with the respective transcript counts and normalized to RPKM (number of reads which map per kilobase of exon model per million mapped reads (Mortazavi et al, 2008)).
high throughput sequence alignment protocol
Sequence reads were aligned to the mm9 reference mouse genome sequence (Waterston et al, 2002) using bowtie (Langmead et al, 2009). Sequence reads that were not aligned to the genomic sequence were aligned to a database of all possible exon-exon junction sequences of the RefSeq transcript deposit.
nucleic acid sequencing protocol
Barcoded RNA-Seq libraries were clustered on the cBot using Truseq SR cluster kit v2.5. Fifty plus bp single end reads were obtained on the Illumina HiSeq2000 using Truseq SBS kit-HS 50 bp (Illumina).
For obtaining cells for the following RNA-Seq experiments, 4 x 10^4 L-K+S+IL7Ra-CD34+FcgRII/III+ cells were preparatively sorted using a FACSAria cell sorter (BD).
nucleic acid library construction protocol
The TruSeq DNA Sample PrepKit (Illumina) was used and the resulting cDNAs were fragmented using AFA technology (Covaris). Subsequently, cDNA fragments were end repaired and 3' adenylated. Illuminas TruSeq adaptors with integrated indices were ligated and the resultant fragments size selected using 2% E-Gels (Invitrogen). Enrichment was done by linear PCR and the resulting fragments were purified using Agencourt AMPure XP magnetic beads.
nucleic acid extraction protocol
RNA was extracted with the Pico Pure RNA Extraction Kit (Arcturus) according to the manufacturers instructions. The resulting RNA was analysed using an Agilent Bioanalyzer. RNA samples were amplified with Nugen`s Ovation RNA-Seq System and quantified by Qubit measurement.
5 x 105 BM cells from R26/AE-reconstituted C57BL/6 recipients that had been exposed to DOX for 18 month were injected into secondary C57BL/6 recipients (2 x 5.5 Gy) and the DOX regime was continued for an additional four month
C57BL/6 recipients (2 x 5.5 Gy) were injected with 5 x 105 non-induced R26/AE BM cells and exposed to DOX for ten days
Non-induced compound R26/AE controls