GSM9289442: Ind3_CX_2.5_48_3; Homo sapiens; RNA-Seq
Frozen iPSC-derived cardiomyocyte pellets were thawed on ice and lysed in Buffer RLT supplemented with 1% β-mercaptoethanol, prepared fresh under a fume hood. Lysates were homogenized using QIAshredder columns (Qiagen; 79656) and mixed with 70% ethanol prior to binding to RNeasy Mini spin columns (Qiagen; 74106). On-column DNase I digestion (Qiagen; 79256) was performed using freshly prepared DNase/RDD buffer mix and incubated for 15 minutes at room temperature. Columns were washed sequentially with RW1 and RPE buffers, and RNA was eluted in 30 µL RNase-free water. All centrifugation steps were carried out at >10,000 rpm as recommended. Eluted RNA was immediately placed on ice and stored at –80 °C. RNA integrity and concentration were assessed prior to library preparation. Polyadenylated mRNA was isolated from 150 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490L; New England Biolabs). Strand-specific RNA-seq libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7765S; NEB), with dual indexing performed using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, E6440S). Libraries were prepared in two balanced batches, each containing all treatment conditions and timepoints for three individuals (Batch 1: Individuals 3; Batch 2: Individuals 1). Final library quality and size distribution were evaluated using the Agilent 2100 Bioanalyzer prior to sequencing.