GSM8516686: SCC25, p63 enhancer library, replicate 4, RNA; Homo sapiens; OTHER
Total RNA was extracted (Quick RNA, Zymo, #R1055). 30 µg per sample of poly-adenylated mRNA was isolated using oligo d(T) beads (NEB E7490L). Resulting mRNA was split into 6 separate reactions and cDNA was synthesized using a gene-specific primer (SL1972) and MultiScribe Reverse Transcriptase (Invitrogen, 4311235) . Following cDNA synthesis, all cDNA samples were pooled and one junction-PCR reaction was performed with 16 cycles (SL1973, SL1974) and I5 and i7 primers from NEBNext Oligo Kit (E7600S) were used for Illumina barcoding with between 5 and 9 cycles. MPRA plasmid DNA pools were amplified with i5 and i7 primers as a control for oligo representation. All libraries were pooled and sequenced as single-end, 100bp on an Illumina NextSeq 2000 instrument at the University at Albany Center for Functional Genomics.