Transcriptomes of NGN2-induced iPSC-derived Neurons

To obtain iPSCs (source: KYOU-DXR0109B) expressing the TetON system and NGN2 gene, rtTA-N144 and TRET-hNgn2-UBC-PuRo plasmids from Addgene were used. rtTA-N144 was gifted by Andrew Yoo (Addgene plasmid #66810), and pLV_TRET_hNgn2_UBC_Puro by Ron Weiss (Addgene plasmid #61474). Lentiviruses were produced in HEK293T cells (1 million cells in a 6-cm dish) by co-transfection with three helper plasmids (pLP1, pLP2, pVSVG), using Lipofectamine2000 reagent in OPTI-MEM medium. Cells were transfected for 4 hours, then 4 ml of complete growth medium was added. Lentivirus-containing media were harvested 48 hours later, centrifuged, and sterilized through a 0.45 um filter. On day 0, iPSC-TetON-NGN2 were plated on Matrigel-coated dishes at 3x10^4 cells/cm^2 in mTeSR1 medium with Y-27632 (5 uM). Doxycycline (1 ug/ml) was added from day 0 to day 5 to induce NGN2 expression, with daily medium changes. Ara-C (0.1 ug/ml) was added on days 2 and 3 to stop iPSC proliferation. New dishes were coated with poly-D-lysine and Matrigel. On day 4, cultures were treated with Accutase, washed, and replated in new dishes pre-coated with poly-D-lysine and Matrigel in N2B27 and mTeSR1 (1:1) medium with BDNF (10 ng/ml), NGF (20 ng/ml), Y-27632 (5 uM), and doxycycline (2 ug/ml). On day 5, the medium was switched to N2B27 with BDNF, NGF, Y-27632, and doxycycline (1 ug/ml). From day 7, half the medium was changed twice a week without doxycycline. Cells were harvested by Accutase cell detachment solution (Stem Cell Technologies, Canada) washed with DPBS and collected by centrifugation. Total RNA was extracted using the ExtractRNA kit (Evrogen, Russia), according to the manufacturer's instructions. cDNA libraries were made starting with 1 ug of RNA with NEBNext Ultra II Directional RNA Library Prep (New England Biolabs, USA) and barcoded with index primers for pooled sequencing.