title: GSM8189759: NSC, TF, RNA, rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Briefly, RNA was isolated with Trizol (Sigma), following manufacturer's instruction and split into two replicates. The mRNA fraction was captured from each replicate of total RNA using Oligo (dT) 25 beads (61002, Life Technologies), DNaseI treated (18068-015, Life Technologies), and reverse transcribed with a GFP-mRNA specific primer (149 STARRseq rep RNA cDNA synth) following Superscript III protocol (18080-044, Life Technologies). To avoid plasmid DNA contamination, cDNA was PCR amplified in 10 parallel reactions for each replicate using primers spanning a synthetic intron (152 STARR reporter specific primer 2 fw and 153 STARR reporter specific primer 2 rv), as previously described (Arnold et al., 2013). 35 ng of PCR amplified DNA were then used in a second round of PCR to add Illumina index primers (E7335, E7500, NEB). PCR was performed in 5 parallel reactions and using 2 independent index primers for each of the two replicates. In total, we sequenced 4 replicates of STARR-seq RNA for each pooled HIST or TF plasmid library. Corresponding plasmid libraries were similarly amplified using two independent Illumina index primers for each plasmid library. Quantity and quality of generated sequencing libraries was assessed on an Agilent Tapestation. All sequencing occurred on an Illumina HiSeq 2500 platform, using 50bp paired-end sequencing.