GSM7663692: 20-637-062_hom_D6; Homo sapiens; RNA-Seq
RNA was isolated from hiPSC-derived iNs at days 0, 2, 6, 13, and 23 using the AllPrep DNA/RNA mini kit (Qiagen). Samples were extracted manually according to the manufacturer's instructions or on a QIAcube using the AllPrep DNA/RNA mini kit protocol (Qiagen). For each timepoint, RNA from 4 technical replicates (4 independent wells of cells cultured simultaneously) were used per line. RNA library preparation from total RNA was conducted following the manufacturer's protocol for the Kapa mRNA HyperPrep Kit (Roche). Briefly, 250 ng of total RNA was enriched for mRNA using magnetic oligo-dT beads. The remaining RNA was then fragmented by magnesium under elevated temperature. After fragmentation, RNA was depleted of rRNA and globin mRNA using the QIAseq FastSelect RNA Removal Kit (Qiagen). The depleted RNA then underwent first strand synthesis using reverse transcriptase and random primers. Combined second strand synthesis and A-tailing incorporated dUTP into the second cDNA strand for stranded RNA sequencing and added dAMP to the 3' ends for adapter ligation. The cDNA fragments were then ligated to sequencing adaptors (IDT xGEN Dual Index UMI adapters) and was enriched using 16 cycles of PCR. Final libraries were assessed using the Agilent Tapestation and Qubit (ThermoFisher) assay methods then sequenced on an Illumina HiSeq 4000 sequencer using 2 x 75bp read length.