GSM8075864: iPSC-derived motor neuron cultures, control, line EDi043-A, Chip, Flow, rep 2, experiment 5.3; Homo sapiens; RNA-Seq
The SMART-Seq V4 Ultra Low RNA Input Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA) was used for reverse transcription and generation of double stranded cDNA for subsequent library preparation using the Nextera XT Library Preparation kit (Illumina, San Diego, CA).  An input of 10 ng RNA was used for oligo(dT)-primed reverse transcription, followed by cDNA amplification and cleanup.  Quantification of cDNA was performed using Qubit (Thermo Fisher Scientific).  cDNA normalized to 80 pg/l was fragmented and sequencing primers added simultaneously.  A limiting-cycle PCR added Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for cluster formation on the sequencing flow cell.  Indexed libraries were pooled and cleaned up, and the pooled library size was verified on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and quantified via Qubit.  Libraries were sequenced on a NovaSeq 6000 (Illumina) using with a 1x75 bp read length and coverage of over 30M reads/sample. The top channel of chip samples or a well from a 96 well plate were incubated with cold Accutase (STEMCELL Technologies) for 20 minutes at 37°C, then cells were extracted by flushing and washing with cold phosphate buffered saline (PBS), pelleted by centrifuge at 350 rcf, and frozen at -80°C. RNA was extracted from pellets using the RNeasy Micro Kit (Qiagen). Various methods:  see SAMPLES "library construction protocol" section