GSM3669336: Clone 1_1; Homo sapiens; RNA-Seq
Total RNA was harvested from a 100% confluent 10-cm plate with 1 mL Trizol and stored at −80 °C. Samples were thawed, homogenized with a 25-gauge needle, and 200 μL chloroform was added. The samples were vortexed (20 s), centrifuged (15,000 g, 30 min), and the aqueous phase was isolated for purification. The aqueous phase was combined with 1 volume of isopropanol, centrifuged (15,000 g, 60 min), and washed with 70% ethanol. The resulting RNA pellet was air dried for 1 h and resuspended in 30 μL RNase-free water. RNA quality was assessed on an Agilent 2100 Bioanalyzer to ensure RIN values were greater than 8.0. For library prep, we used the NEBNext Ultra Directional Library Prep Kit (New England Biolabs). Briefly, 500 ng of RNA in 50 μL water was combined with 15 μL NEBNext Oligo d(T)25 magnetic beads (pre-washed twice with RNA binding buffer) and incubated at 65 °C for 5 min. The beads were washed twice with 200 μL wash buffer and eluted with 200 μL elution buffer. The binding-washing was repeated once more. The final elution was in 15 μL first strand synthesis buffer and incubated at 94 °C for 15 min. Then 10 μL of the supernatant was used for cDNA synthesis reaction, which was purified using Agencourt AMPure XP beads. End-repair was performed with NEBNext end repair reaction buffer and purified with AMPure XP beads. Library enrichment and multiplexing was performed using the NEBNext High-Fidelty PCR Master Mix (14 cycles of PCR) and NEBNext Multiplex Oligos. The cDNA libraries were multiplexed on the Illumina HiSeq 2000.