GSM8223164: TSP77 LOF rep2; Homo sapiens; RNA-Seq
NPCs were collected and pelleted at day 10 of differentiation. Pellets were stored at -80°C until all samples had been collected. All samples RNA were extracted at the same time. RNA was extracted following the protocol in RNeasy Mini Kit (Qiagen). RNA extraction inlcuded treatment with DNase I following the optional portion of the RNeasy kit using the DNase I kit (Qiagen). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation.  Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags.