GSM8796835: STARR-seq output-naive, biological replicate 4; Homo sapiens; OTHER
Total RNA was extracted from transfected ARPE-19 cells using M5 HiPer Universal RNA Mini Kit (Mei5bio), and followed by poly (A) tail mRNA isolation using Dynabeads® Oligo (dT) kit (ThermoFisher Scientific). mRNA was treated with TURBO™ DNase (ThermoFisher Scientific) and purified by Agencourt RNAClean XP beads (Beckman Coulter). Target-specific first strand complementary DNA (cDNA) synthesis was performed with SuperScript III (ThermoFisher Scientific; 1.5 μg mRNA/reaction) to specifically reverse transcribe plasmid transcribed mRNAs (RT primer: 5'- CAAACTCATCAATGTATCTTATCATG-3'). All mRNA from the 10 million transfected cells was used in reverse transcription. After reverse transcription, reactions were treated with RNase A + H and pooled. Then the first round of amplification, every 5 μL cDNA reaction was used as a template in every 50 μL PCR reaction using KAPA HiFi HotStart ReadyMix (Roche). Target amplification primers were used (gia-starrseq-2-f: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCTattagattgatctagagcatgcaccggt-3'; gia-starrseq-2-r: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCtcgaagcggccggccgaattcgtcga-3'). In the final round of amplification, the enzyme used is the same as that used in the first round, but 4 sets of primer combinations with different indexes are set to complete the final construction of the library. Illumina HiSeq platform compatible primers were used (forward: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; reverse: 5'-CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTG-3'). A total of 4 duplicated PCR reactions were performed for each indexing PCR. PCR products were cleaned up by DNA pure beads (VDOBIOTECH) and submitted for Illumina sequencing. Input library for sequencing was generated using similar PCR setup. Products from 4 duplicated PCR reactions were pooled (10 ng plasmid/reaction, 16 cycles of PCR).