V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for one passage off of MEFs, on gelatinized tissue-culture plates. Cells harbouring a Dox-repressible Oct4 allele (ZHBT-c4 cells) were treated with 2mg/ml doxycycline (SIGMA, D-9891) for 12hrs or 24hrs. To generate neural precursor cells, ES cells were differentiated along the neural lineage using standard protocols. V6.5 ES cells were differentiated into neural progenitor cells (NPCs) through embryoid body formation for 4 days and selection in ITSFn media for 5–7 days, and maintained in FGF2 and EGF2 (R&D Systems). Mouse embryonic fibroblasts were prepared and cultured from DR-4 strain mice.
Extracted molecule
total RNA
Extraction protocol
For miRNA, Single-stranded cDNA libraries of short transcripts were generated using size selected RNA. RNA extraction was performed using Trizol, followed by RNeasy purification (Qiagen). 5µg of RNA was size selected and gel purified. 3’ Adaptor was ligated to RNA with T4 RNA ligase and also, separately with RNA Ligase. Ligation products were gel purified and mixed. 5’ adaptor was ligated with T4 RNA Ligase. RT-PCR (Superscript II, Invitrogen) was performed with 5’ primer. Splicing of overlapping ends PCR (SOEPCR) was performed (Phusion, NEB) with 5’ primer and 3’ PCR primer, generating cDNA with extended 3’ adaptor sequence. PCR product was denatured and the differently sized strands were purified.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
other
Instrument model
Illumina Genome Analyzer
Description
ZHBT-c4 cells were described in Niwa et al., Nature Genetics 2000. They are cultured in standard ES cell medium +/- doxycycline.
Data processing
Images analysis and base calling was done using solexa pipeline and reads aligned to both mouse NCBI build 36 and 37 using ELAND. For miRNA analysis - 5p end defined by start of tag, 3p end defined by 6nt perfect match to 3p linker and . Alignments to build 37 were used for analysis of the mmu-mir-290 cluster only as that cluster is not represented on build 36. For ChIP-Seq, sequences from all lanes were extended 200bp (maximum fragment length accounting for ~100bp of primer sequence), and allocated into 25 bp bins. Genomic bins containing statistically significant ChIP-seq enrichment were identified by comparison to a Poissonian background model, using a p-value threshold of 10-9. Additionally, we used an empirical background model obtained from identical Solexa sequencing of DNA from whole cell extract (WCE) from matched cell samples (>5X normalized enrichment across the entire region).
