title: GSM9574283: RNase H1 D145N-transduced hiPSC-derived neurons, Patient 553, culture 1, RNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted from cells by adding 1 mL TRIzol (Invitrogen, cat. no. # 15596026) directly to the culture plate and scraping vigorously with a pipette tip. RNA was extracted with chloroform and precipitated with isopropyl alcohol, then treated with 2 U/10 μg RNA RNase-free DNase I (Invitrogen, cat. no. #AM222) at 37°C for 30 mins to remove DNA contamination. RNA was cleaned with acid phenol/chloroform and ethanol precipitated. Aliquots were stored at -80° prior to NGS library construction. NGS libraries from total, purified RNA were constructed with the SMARTer Stranded RNA-seq Kit (Takara, cat. no. #634839) according to the manufacturer's instructions for first-strand cDNA synthesis, purification of first-strand cDNA using SPRI beads, RNA-seq library amplification via PCR (with 12 PCR cycles for 10 ng of starting RNA), and SPRI cleanup. All library sizes and concentrations used for molarity calculations were confirmed on an Agilent 2100 Bioanalyzer and a Qubit 4 fluorometer (ThermoFisher Scientific) with a Qubit dsDNA HS Assay kit (ThermoFisher Scientific, cat. no. #Q32854), respectively. RNA-sequencing was performed on an Illumina HiSeq 2500 (single-indexed libraries). All RNA-sequencing (2x75 bp) was seqeunced to a minimum read depth of 50,000,000 reads. RNA-sequencing libraries were split across three lanes and merged for downsream analysis.