GSM7912885: endoSTARR_5bp_plDNA; Homo sapiens; OTHER
Cells were harvested 24h post transfection and lysed in Trizol. RNA was extracted by Chloroform extraction, followed by Isopropanol precipitation with a 1:1 ratio. RNA pellets were resuspended in RNAse-free water and digested with DNAseI for 20 min at room-temperature. DNAseI-treated RNA was purified with a RNA-clean&concentrator-25 kit (Zymo). A total of 10-12 μg RNA was reverse transcribed using the STARR-seq specific RT primer. For each μl of Maxima H minus RT 3 μg input was used. RT reactions were diluted 1:4 with RNAse & DNAse-free water before further amplification. The splice-junction PCR was run using the STARR-seq thio-splice-junction primer and a custom reverse primer spanning the SalI restriction site used for linearizing the STARR-seq vector and the read1 Nextera adapter sequence inserted with the library. About one third of the RT reaction was amplified for 19-22 cycles. Illumina barcodes were added by a final PCR with 8 cycles using the Illumina Nextera guidelines. After each amplification step, PCR products were cleaned up using AMPure XP magenetic beads (Beckman Coulter). Plasmid libraries were directly amplified with a 8-cycle PCR using NEXTERA-specific index primers. For endogenous libraries, cells were grown for ~10-12 days in standard media before FACS sorting for GFP-negative cells. After sorting, library-containing cells were grown for another 6-7 days before RNA and genomicDNA extraction. Extractions were done in triplicates using a Mini Allprep mRNA/gDNA extraction kit (QIagen). A total of 24 μg RNA was reverse transcribed per replicate using the STARR-seq specific RT primer. RT reactions were cleaned up using a Clean&Concentrator kit (Zymo) with 7x Binding Buffer. gDNA and mRNA samples were amplified (20 cycles) using either an intron-specific primer (gDNA) or the standard STARR-seq splice-junction primer (mRNA) together with a custom reverse primer (see above). Illumina barcodes were added by a final PCR with 9 cycles using the Illumina Nextera guidelines. After each amplification step, PCR products were cleaned up using AMPure XP magenetic beads (Beckman Coulter). Plasmid libraries were directly amplified with a 8-cycle PCR using NEXTERA-specific index primers. NEXTERA