Chapter 2 68 SUPPLEMENTARY METHODS Supplementary Table 1. iPSC lines Line Genotype Age Sex Repr. method Used for experiments C1 Control 3d M Lentiviral RNA sequencing C2 Control 74d M Lentiviral All C3 Control 70y M Lentiviral RNA sequencing C4 Control 19y F Lentiviral Neuronal analysis, Neuron-glia coculture, qPCR C5 Control 21y M Lentiviral All except RNA sequencing C6 Control 19y M Lentiviral All except RNA sequencing P1 4H (POLR3B) 10y F Lentiviral All P2 4H (POLR3B) 2y M Lentiviral All P3 4H (POLR3B) 4.5y F Lentiviral All P4 4H (POLR3A) 12y F Lentiviral All except RNA sequencing P5 4H (POLR3A) 24y M Sendai Neuron-glia co-culture, qPCR P6 4H (POLR3A) 2y F Sendai Neuron-glia co-culture, qPCR Repr. method: reprogramming method RNA isolation Total RNA was isolated from samples in Trizol using a chloroform-isopropanol extraction procedure. Briefly, cells were washed 3x with PBS (Braun Medical) and then incubated in 750uL Trizol for < 2 min at room temperature (RT). Samples in Trizol were transferred to 1.5mL Eppendorf tubes (VWR), dissolving any clumps with gentle titration, and stored at -80 C until needed for RNA isolation. After thawing sample tubes at RT, 150uL of chloroform was added to samples in Trizol, solutions mixed by shaking, and tube allowed to stand for 10min at RT. Tubes were then centrifuged (>13K g, 10min, 4C) and the top, clear liquid layer containing RNA transferred to new tubes. RNA was then precipitated by addition of 350uL isopropanol (Sigma-Aldrich) for every 750uL Trizol in the original sample. The tubes were inverted gently to mix, and the solution was allowed to stand for 10min at RT. The samples were centrifuged again (>13K g, 10min, 4C) and the supernatant was removed from the pellet. The pellet was then washed 2x with EtOH (VWR), with centrifugation after each wash step (7500g, 5min, 4C). The pellet was allowed to air-dry before dissolving in appropriate quantity of DMPC water and stored at -80C until needed. RNA concentration and quality were initially measured on a Nanodrop 3000.
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