GSM9257863: NPPO-1nonNE, RNA-seq; Mus musculus; RNA-Seq
Organoids were dissociated into single cells using TrypLE, and total RNA isolated using MagMAX™-96 for Microarrays Total RNA Isolation Kit (ThermoFisher Scientific AM1839). Briefly, cells were lysed in 1 ml TRI reagent per 5 million cells, and incubated for 5 min at RT. The homogenate was mixed with 0.1 volume 1-bromo-3-chloropropane (Millipore-Sigma B9673), incubated at RT for 5 min, and centrifuged at 12,000 g for 10 min at 4°C. 100 μl of the aqueous phase was transferred to an 8-strip EpiCypher tube (DNase/RNase-free), and 50 μl of 100% isopropanol (Millipore-Sigma I9516) added with shaking for 1 min. 10 μl of RNA Binding Beads were added, followed by shaking for 3 minutes. The RNA Binding Beads were then captured on a magnetic stand and the beads washed twice with 150 μl Wash Solution. The beads were dried for 2 min, and RNA eluted in 50 μl of Elution Buffer. All samples were DNase-treated prior to library treatment. Quality assessment was performed using the 4200 TapeStation system (Agilent), with a median RNA integrity number of 9.7 (range 9.5 to 10). Libraries were generated using the Illumina Stranded mRNA Prep, and 150-bp paired-end sequencing was performed to a minimum of ~30 million reads per sample on the Illumina HiSeq platform.