2 protocols
Trimmed sequences in 3' to 36 nts mapped to hg19 with BOWTIE -m40 -v2 assigned counts to gene models in ENS65 with DEGseq Genome_build: hg19 (HeLaS3) or mm9 (germ cells) Supplementary_files_format_and_content: RPKM and count table for ENS65
nucleic acid library construction protocol
RNA was isolated with TRIzol, depleted of polyA+ transcripts with oligo-dT beads, and depleted of rRNA with the Ribominus kit libraries were prepared with the dUTP strand-specific protocol. After RT and second-strand synthesis, 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 T4 ligase (Enzymatics). Fragments of 200±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530) after removal of the 2nd strand with heat-labile UDG (Roche).