GSM8482965: 16HBE cells, 5% patient serum 552590, 24h, rep1; Homo sapiens; RNA-Seq
At end of incubation, media was removed, lysis buffer was added, and cells were stored at -80°C for up to two weeks before RNA isolation. Total RNA was isolated by automated isolation using a MagMax mirVana kit (Invitrogen A27828) on either a KingFisher Flex or a Kingfisher Duo Prime as per the manufacturer's recommendation. RNA was eluted in 100 µL of elution buffer, quantified using a Qubit (Qubit RNA BR Assay Kit), and stored at -80°C. For some iCAP-RNAseq experiments, total RNA was isolated manually using RNeasy Mini Kit (Qiagen). 100-650 ng of total RNA from each iCAP sample was used for automated library preparation and RNA sequencing (RNAseq) performed by Covance or Genewiz (now Azenta life sciences). Strand-specific library prep was performed with PolyA selection using TruSeq RNA library prep kit with unique dial indices (IDT) and resulting DNA was sequenced on an Illumina Hiseq 4000 with paired end 150 bp reads