Cells were grown in Schneider's (FBrf0024118) supplemented with 10% fetal calf serum.
Cells were maintained between 2E6 and 1E7 cells/mL, and were harvested at about 5E6 cells/mL.
Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Oligotex kit, then fragmented using divalent cations under elevated temperature. First and second strand cDNA synthesis was primed with random hexamers. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase, and phosphorylated at their 5' ends with T4 polynucleotide kinase. After adding A bases to the 3' end of the DNA fragments, Illumina adaptor oligonucleotides were ligated to the ends and ~ 300 bp fragments were isolated from an agarose gel, enriched by PCR amplification, and gel-purified again.
Read length (bases):37
The samples were quantitated using a Nanodrop, and loaded onto a flow cell for cluster generation and sequenced on an Illumina Genome Analyzer II using either single read or paired end protocols (Illumina).