GSM8968168: CHyMErA in RPE-1 cells, T21 post infection, TG003 treatment, replicate A; Homo sapiens; OTHER
CRISPR sequencing libraries were prepared as previously described (PMID: 32249828). Briefly, at each timepoint, cells were collected at 100-fold of library coverage, washed in PBS, and stored at -80 until processing. Genomic DNA was extracted using the Wizard™ Genomic DNA Purification Kit (Promega #A1125) according to the manufacturer's protocol. DNA concentration was determined using the Qubit dsDNA Broad Range Assay kit (Invitrogen). For PCR amplification of hgRNA sequences, the following primers were used to amplify the integrated library from genomic DNA at >95-fold coverage, using NEBNext Ultra II Q5 Master Mix (NEB #M0544L). PCR1_Hybrid_Outer_F2: TGAAGAATCGCAAAACCAGCA PCR1_Hybrid_Outer_R1: GAGGGCCTATTTCCCATGATTC The resulting products were analyzed by gel electrophoresis for size and quality. PCR products were then pooled and subjected to indexing by PCR using Illumina TruSeq i5 and i7 barcoded adapters. Sequencing libraries were analyzed by agarose gel electrophoresis. Gel extraction was performed for the correct PCR product using PureLink™ PCR Purification Kit (Invitrogen, #K310001).Sequencing libraries were submitted for quality control analysis and high-throughput sequencing at the Donnelly Sequencing Centre (DSC). 1 μl top stock of each purified library was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA). The libraries were quantified using the Quant-iT dsDNA high-sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA) and were pooled at equimolar ratios after size-adjustment. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (cat # E7630L, New England Biolabs, Ipswich, USA). The quantified pool was hybridized at a final concentration of 350 pM and sequenced at a depth of 50 M reads/sample on the Illumina NovaSeq 6000 platform using a SP flow cell of the following parameters: DC1: 29bp, R1: 33bp, IR1: 8bp, IR2: 8bp, DC2:20bp, R2: 32bp.