title: GSM8191374: ZIC3 KO_C2, short-read RNA-seq, biol rep 1 of 1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol (Invitrogen) followed by column purification with the RNeasy MinElute Cleanup kit (Qiagen). Short-read RNA-seq was completed by the Indiana University School of Medicine Center for Molecular Genomics. Total RNA was extracted from wildtype, ZIC3 AtoG_C1, ZIC3 AtoG_C2, ZIC3 KO_C1, and ZIC3 KO_C2 H1-OCT4-EGFP cells. The RNA integrity number (RIN) was determined using an Agilent TapeStation (Agilent Technologies) followed by an RNA purification step with the KAPA RNA HyperPrep kit (Roche). cDNA libraries were prepared using the NovaSeq 6000 SP Reagent kit v1.5 (300 cycles) and sequenced using the NovaSeq 6000 (Illumina) with 2 x 150 bp paired-end reads.   Long-read RNA-seq was performed by the Genome Access Technology Center at Washington University in St. Louis. Total RNA was extracted from control (ZIC3 WT) (n=3), ZIC3 AtoG_C1 (n=3), and ZIC3 KO_C1 (n=3) H1-OCT4-EGFP cells and the RIN determined with an Agilent Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared with the PCR-cDNA Barcoding kit SQK-PCB111.24 (Oxford Nanopore Technologies) and the Agilent Bioanalyzer (Agilent Technologies) served for quality assessment. Cell samples were pooled together on three PromethION R9.4.1 flow cells (50 fM/flow cell) and sequenced in a PromethION 24 A100 (Oxford Nanopore Technologies) for 90 h.