title: GSM2171428: Archer_T47D_RNASeq_Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Start-Seq: Start-Seq experiments were performed as previously described (Nechaev et al., Science 2010). RNA-Seq: Total RNA was harvested using an RNeasy kit (Qiagen) with on-column DNase treatment.  RNA quality was validated by Bioanalyzer (Agilent). Paired-end strand-specific poly-A enriched RNA-seq libraries were generated by the NIH Intramural Sequencing Center (NISC). MNase-Seq: Nuclei were harvested from T-47D cells and digested for 5 minutes at 37C with a range of MNase (Worthington) concentrations. Reactions were stopped by addition of EDTA and then treated with RNase and proteinase K. Digested DNA was isolated by phenol/chloroform extraction and ethanol precipitation. FAIRE-Seq: FAIRE-seq experiments were performed as previously described (Burd et al., Mol Cell Biol 2012). Start-Seq: Libraries were constructed as previously described (Nechaev et al., Science 2010) using an Illumina TruSeq small RNA kit. RNA-Seq: Libraries were constructed using an Illumina TruSeq Stranded mRNA kit. MNase-Seq: Libraries were constructed using an Illumina TruSeq sample preparation kit. FAIRE-Seq: Libraries were constructed as previously described (Burd et al., Mol Cell Biol 2012). RNA-Seq: Prior to alignment, RNA-Seq reads were filtered based on quality; reads with an average Sanger quality score less than 20 were removed from analysis. Following quality filtering, Cutadapt was used to remove adapter sequences. Insert lengths were estimated by transcriptome alignment using Bowtie. Sequence alignment was then performed using TopHat (version 2.0.4) [35]. Following de-duplication by Picard, alignments from individual Replicates were merged. FPKM values were calculated using Cufflinks (version 2.2.1).