3 protocols
normalization data transformation protocol
reads were trimmed to 30 nts mapped to mm9 with BOWTIE -m40 -v2 --best reads were assigned to gene model in ENSEMBL v67 using the R package DEG-seq Genome_build: mm9 Supplementary_files_format_and_content: Table of raw RNA-seq counts and normalized RPKM values for all genes models in ENSEMBL v67. The table is linked as a supplementary file on the Series record.
nucleic acid library construction protocol
Cells were lysed in TRIzol and RNA extracted with standard procedures. PolyA selection was performed with two rounds of purification on oligodT(25) magnetic beads. RNA was fragmented by boiling in presence of divalent cations. RT was performed with random hexamers and dUTP was incorporated in the 2nd strand to retain strand information. Libraries were prepared according to manufacturer's instructions (Illumina). cDNA was end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, #M8225). Fragments of 300±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530) after treatment with UDG to remove the second strand.
growth protocol
E14 cells were grown in standard ESC conditions including 15% ESC certified serum