GSM8973811: RNA, HeLa cells transfected with 500 ng DNA plasmid pool 3; Homo sapiens; OTHER
Plasmid pools were isolated from NEB 10-beta electrocompetent E. coli using a QIAGEN Plasmid Purification Kit (total of 3 DNA plasmid pools). RNA was extracted using TRIzol from HeLa cells transfected with different amounts of each plasmid pool (total of 9 RNA samples). DNase-treated RNA was reverse transcribed using SuperScript IV VILO (Thermo Fisher Scientific, #11756050) which includes both, oligo(dT) and random primers.  Both, the resulting cDNA (=expression) and the original plasmid DNA pool that was transfected (=input) was amplified with Q5 polymerase (NEB, #M0491L) in a nested PCR: first with primers that bind to MusD6 and that have an overhang complementarity to the Illumina sequencing primers (MPRA_#3 5'-ACACGACGCTCTTCCGATCTccttctcagtcgaaccgttc-3', MPRA_#4 5'-GACGTGTGCTCTTCCGATCTcatgtgtacctgtccgttgc-3'), then in a second PCR with Illumina-P5-universal-for and Illumina-P7-index-rev primers. After clean-up and pooling, RNA-libraries (expression) and DNA-libraries (input plasmid pools) were sequenced on an Illumina NextSeq500 (SE150) with ~50% phiX spike in.