GSM6030628: pDNA_4_S28; Homo sapiens; OTHER
~15-20 x106 cells were collected per sample and treated with 100µg of cycloheximide on ice for 10 minutes. Cells were lysed and for each sample, ~10% saved for plasmid DNA isolation, ~25% saved for total mRNA isolation, and ~65% loaded onto a sucrose gradient for polysome mRNA isolation. Plasmid DNA was isolated using Qiagen Spin Miniprep Kit. Total mRNA and polysome mRNA was isolated using Zymo Direct-zol RNA Miniprep Plus kit with DNase I treatment. cDNA was made using SuperScript III first-strand synthesis system and RT primer: 5'- GGTTTGTCCAAACTCATCAATGT -3'. Library construction was performed using a two-step PCR amplicon approach. First amplification was performed at 98°C for 30” followed by ten cycles of 98°C for 10”, 71°C for 30”, 72°C for 30”, and final elongation at 72°C for 5' with Forward primer: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGgccagcgccaggatcaac -3' and Reverse Primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcgccccgactctagctagac -3' using Q5 Polymerase. PCR product was purified using 1.8X Ampure XP beads. Second amplification was was performed at 98°C for 30” followed by ten cycles of 98°C for 10”, 67°C for 30”, 72°C for 30”, and final elongation at 72°C for 5' with IDT for Illumina Nextera UDI primers using Q5 Polymerase. PCR product was purified using 0.7X Ampure XP beads.