GSM8524936: nasal-organoids, HNO918line, differentiated, day21, 37Ctreated, rep2; Homo sapiens; RNA-Seq
Total cellular RNAs were extracted from nasal organods using RNeasy Mini Kit (Qiagen). The RNA integrity was assessed on a 2100 Bioanalyzer (Agilent). A double-stranded cDNA library was created using 500 ng of total RNA (measured by picogreen), preparing the fragments for hybridization onto a flowcell. An ERCC RNA Spike-In Control Mix 1 was added to each sample according to the manufacturer's protocol. First, cytoplasmic and mitochondrial ribosomal RNA was removed from the total RNA samples using Illumina's Epidemiology-Specific RiboZero rRNA removal protocol. Then cDNA was generated using the fragmented and rRNA-depleted total RNA using random primers. During second-strand synthesis, deoxythymidine triphosphate (dTTP) was replaced with deoxyuridine triphosphate (dUTP) which quenches the second strand during amplification, thereby achieving strand specificity. Libraries were created from the cDNA by first blunt ending the fragments, attaching an adenosine to the 3′ end, and finally ligating unique adapters to the ends for PCR amplification. Paired end sequencing (2 × 150 bp) was performed on an Illumina HiSeq 4000 for a depth of 30 million paired end reads (a total of 60 million reads) per RNA sample.