GSM7316662: Synthetic_Library; Homo sapiens; RNA-Seq
Twenty-four hours post-transfection, cells were pelleted and washed with DPBS. Total RNA was extracted using Trizol (Invitrogen, 12183555). From 75 μg of total RNA per sample, mRNAs were isolated using the Dynabeads mRNA Purification Kit (Invitrogen, 61006). Residual DNA was removed from 1 μg of mRNA using ezDNase (Invitrogen, 11766051). All mRNAs were subjected to a reverse-transcription reaction in 8-strip PCR tubes, using a custom primer（R-amp1-UMI12-rd2, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNggccgtttacgtcgccgtcc， FS-TSO, AAGCAGTGGTATCAACGCAGAGTACrGrGrG） and the SuperScript IV First Strand Synthesis Kit (Invitrogen, 18091050). The resulting cDNA was purified using the QIAquick PCR Purification Kit (Qiagen, 28506). The purified cDNA was split into 8 PCR tubes and amplified using NEBNext Ultra II Q5 Master Mix (NEB, M0544S) and F-TSO(ISPCR)-rd1( ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCAGTGGTATCAACGCAGAG) and P7-i7(index)-rd2(CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) primers for three cycles. After the PCR reaction, all eight tubes for each sample were combined in a DNA LoBind tube. PCR products were purified using AMPure XP (Beckman Coulter, A63881) and then further amplified using P5-i5-rd1(AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT) and P7(CAAGCAGAAGACGGCATACGAGAT) primers for seven PCR cycles to add P5, P7, and unique 8 bp Illumina index sequences. Similarly, plasmid DNA was amplified in 2x50 μL reactions using NGS-F and NGS-R primers and amplified using NGS-P5 (ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGATAACCGTATTACCGCCATGC), and NGS-P7(GACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGAACAGCTCCTCGCCCTTG) primers for seven PCR cycles to add P5, P7. Following SPRI (Beckman Coulter, B23319) cleanup, amplicons were sequenced using a NovaSeq 6000 SP flow cell with a 15% PhiX spike-in.