GSM5454438: STARR-seq HepG2 genomic, replicate 2; Homo sapiens; OTHER
Total RNA was isolated Qiagen RNeasy maxi kit after 24 hours of transfection followed by polyA(+) purification using direct mRNA purfication beads (ThermoFisher). STARR-seq reporter and input library was constructed with minor modifications as described in Arnold et al. Science 2013. First strand cDNA synthesis was done with 2.5-5 ug of polyA(+) RNA and with Superscript III (Invitrogen, #18080-044) using a reporter-RNA specific primer. This was followed by RNase A treatment for 1 h at 37˚C and purification. cDNA amplification was performed with reporter-specific nested cDNA primers corresponding to the respective STARR-seq reporter construct using KAPA HiFi PCR Master mix (Roche) in the same number of reactions as done for the reverse transcription. The final PCR reactions to produce Illumina-compatible sequencing libraries were prepared from the entire amplified cDNA using KAPA HiFi PCR Master mix (Roche) as descriubed in Arnold et al. Science 2013. All purifications were done using AMPure XP beads as per manufacturer's instruction unless otherwise mentioned. Illumina sequencing strategy adapted for CpG-free custom adapters, sequencing and index primers.