GSM8887671: 8p WT cell, mRNA, biological replicate 3; Homo sapiens; RNA-Seq
Total mRNA:Cells from the chromosome 3 isogenic aneuploidy system (3p loss/3q gain/WT) were washed three times with PBS, snap-frozen, and then lysed with the QIAShredder columns (#79654, QIAGEN) according to the manufacturer's instructions. For each cell line, three biological replicates were processed. Total RNA was isolated with the Qiagen RNeasy Mini Kit (#74104, QIAGEN), including the DNA digestion step using the RNAase-Free DNAase set kit (#79254, QIAGEN). Ribosome Profiling:3p loss cells and 3p WT cells were subjected to ribosome profiling. For each cell line, three biological replicates were processed. Cells were cultured under standard conditions until they reached 80% confluency. They were then treated with 10 µg/µL cycloheximide for 5 minutes at 37°C. Following treatment, cells were harvested and lysed. Ribosome-protected fragments (RPFs) were generated and purified using RiboLace Pro (Immagina Biotech; catalog # RL00P-12). The RPFs underwent end repair using the Small RNA Library Prep Kit (NEB; catalog # E7300S) and size selection using the RNA Clean & Concentrator-5 Kit (Zymo; catalog # R1015) to enrich RNA fragments sized smaller than 200 nt. Total mRNA:PolyA isolation and sequencing library preparation was performed using the NEB Next Ultra Directional RNA Library Prep Kit (#E7760, NEB). Each set of samples were pooled and sequenced in one lane of HiSeq2500 RR, 100 basepair paired end. For the transcriptome analysis of chromosome 8 isogenic aneuploidy system (8p loss/8p WT), total mRNA samples were prepared as described above with three biological replicates, and the following library preparation was conducted in Yale Center for Genomic Analysis (YCGA). Sequencing was conducted on NovaSeq X Plus platform (Illumina). Ribosome Profiling:RPFs were reverse transcribed and tagged by Small RNA Library Prep Kit (NEB; catalog # E7300S) following the recommended conditions. RPF libraries were then amplified by Phusion High-Fidelity PCR Kit (NEB; E0553L). Further size selection and purification of RPF libraries was conducted using Mag-Bind TotalPure NGS beads (Omega Bio-tek; catalog # M1378-01) at a beads-to-product ratio of 1.3:1. The NGS libraries were quantified using the sparQ Universal Library Quant Kit (Quantabio; catalog # 95210-100) and analyzed using a TapeStation (Agilent) before being sequenced with NovaSeq X Plus (Illumina) in YCGA or NextSeq 1000 system in Quintara Biosciences. The target sequencing depth for the RPF libraries was 200 million reads per library.