normalization data transformation protocol
Final gene expression quantitation was done with Cufflinks 1.3.0, Ensembl 66 GTF, additional parameter --max-mle-iterations 10000.
high throughput sequence alignment protocol
Reads were trimmed to 70bp due to quality and aligned to mm9 with Tophat 1.3.1 / Bowtie 0.12.7, using the Ensembl 63 GTF file for gene models. Parameters were -g 1 --mate-inner-dist 200 --mate-std-dev 70 --segment-length 35 --segment-mismatches 2; this allows for 4 mismatches per read (two per read half) and unique alignments only.
nucleic acid sequencing protocol
A total of 10 fmol library fragments were loaded to cBot to generate clusters, followed by sequencing on an Illumina HiSeq 2000 to produce 10-30 million paired-end 100 bp reads per sample.
nucleic acid extraction protocol
Illumina TruSeq RNA Sample Prep Kit (Catalog #: FC-122-1001)
All mice used in this study were housed in the animal facility at the Stowers Institute for Medical Research (SIMR) and handled according to SIMR and National Institutes of Health (NIH) guidelines. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of SIMR. H19-DMR flDMR/flDMR mice on B6 background were kindly provided by Dr. Marisa S. Bartolomei (University of Pennsylvania Perelman School of Medicine). Conditional mutant Igf1rfl/+ was kindly provided by Thomas L Clemens (Center for Musculoskeletal Research, Johns Hopkins Medicine, 601 North Caroline Street, Baltimore, MD 21287). Interferon inducible Mx1-Cre or tamoxifen inducible Scl-Cre mouse strains were used to delete the floxed H19-DMR and Igf1r. For Mx1-Cre activation, 250ug of pIpC was injected intraperitoneal every other day for 14 days at 5 weeks of age. For Scl-CreER activation, 2mg tamoxifen dissolved in 0.1 ml of corn oil was injected intraperitoneal every day for 5 days.
nucleic acid library construction protocol
The RNA-sequencing library was prepared from approximately 200 ng of total RNA (mH19dDMR/+ Igf1r-/- , mH19dDMR/+Igf1rf-/- and mH19flDMR/+) for each sample. The fragment size in the generated library ranged from 220 to 500 bps with a peak at 280 bps.