GSM9219921: HeLa MPRA replicate 1; Homo sapiens; RNA-Seq
Total RNA was extracted from cells 36–48 h after transfection using the Monarch RNA cleanup kit (NEB). Poly(A)+ mRNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). mRNA was reverse transcribed with SuperScript IV Reverse Transcriptase (Thermo Fisher) to generate cDNA using a primer downstream of the barcode region. cDNA was amplified with KAPA HiFi HotStart ReadyMix using primers flanking the Citrine exon 1 splice junction and barcode region, which included offset sequences to increase base diversity. Following amplification, Illumina P5/P7 adapters were added via qPCR to selectively enrich for adapter-containing fragments and prevent over-amplification. Libraries were size-selected to retain fragments ≥100 nt, quantified with Qubit and qPCR, pooled with PhiX, and sequenced on an Illumina NextSeq 500 using 300-cycle kits and custom primers.