GSM9541928: HDR_GFP_low_high_induction; Homo sapiens; OTHER
Three days post-nucleofection, a baseline 'total population' of the HEK293-pMM490-Low cell pools was collected, while the remaining cells were sorted by fluorescence-activated cell sorting (FACS) into two populations: GFPconsitutive (cells exhibiting constitutive GFP expression irrespective of risdiplam treatment) and GFP low population (cells with low basal GFP expression). The GFP low population population (1.2 million cells collected) was cultured further and subsequently treated with 150 nM risdiplam for 48 hours to induce splicing, alongside parental HEK293-pMM490 which served as a positive control for FACS gating setup. Following this treatment, the cells were subjected to a second round of FACS and fractionated based on their induced GFP intensity into three distinct populations: 'no response' (1.5 million cells), 'medium response' (0.5 million cells), and 'strong response' (0.8 million cells). All sorted sub-populations, the initial total population, and the parental control were expanded to over 2 million cells. Finally, genomic DNA (gDNA) was extracted from these expanded populations using the DNeasy Blood & Tissue Kit (Qiagen, 69504) according to the manufacturer's instructions. Following extraction, the isolated genomic DNA was frozen and stored at -20°C. The concentration of the extracted double-stranded DNA was quantified using Lumiprobe Pico488, and DNA integrity was assessed using a QIAxcel Fragment Analyzer prior to downstream target amplification. For sequencing library preparation, 3.2 µg of genomic DNA per sample was used as the template for a customized two-step PCR protocol. To maintain library complexity, the first-step PCR consisted of 32 individual reactions per sample (20 µL volume containing 5 µL of template). The mutated region of the SMN2-GFP reporter was specifically targeted using forward primer MM61 (5'-AGCTTTCATGCTATCTTAACTGC-3') and reverse primer MM50 (5'-ACCTTTCAACTTTCTAACATCTG-3'). This primer pair incorporated partial Illumina adapter sequences (tails) and was designed to yield a ~400 bp amplicon while minimizing amplification of the larger corresponding region from the endogenous SMN2 locus. Following this initial amplification, the 32 reaction products for each sample were pooled and purified. The purified pool subsequently served as the template for a single, second-step PCR per sample to complete library construction by adding full Illumina adapters and indices. Final libraries were sequenced on an Illumina NovaSeq platform using an SP flow cell with 2x250 bp paired-end reads.