C1000 touch cycler

Mice used in this study included Gli1-Cre^ERT2 knock-in (JAX#007913), ROSA26^{loxp-STOP-loxp-tdTomato} conditional reporter (JAX#007905, (Madisen et al., 2010 (link)), Gli1-LacZ heterozygotes (JAX#008211, (Bai et al., 2002 (link)), Sox2-Cre^ERT2 knock-in (JAX#017593, (Li et al., 2015 (link)), Runx2-rtTA (gift from Fanxin Long, Washington University School of Medicine; (Chen et al., 2014 (link)), tetO-Cre (JAX#006234), C57BL/6J (JAX#000664), and Runx2^flox/flox (gift from Dr. Takeshi Takarada, Okayama University, Japan; (Takarada et al., 2013 (link)). Mice were housed in pathogen-free conditions and analyzed in a mixed background. All mice were used for analysis without consideration of sex. All mice were induced at one month of age and euthanized at specific stage as described in figure legend. Genotyping was performed from ear biopsies, which were lysed in DirectPCR tail solution (Viagen 102 T) through incubation at 55°C overnight followed by 85°C heat inactivation for 30 min. PCR-based genotyping (GoTaq Green MasterMix, Promega, and C1000Touch Cycler, Bio-rad) was conducted to identify the mouse lines. All mouse experiments were approved by the Institutional Animal Care and Use Committee at the University of Southern California.

Total RNA was extracted using RNeasy Plus Mini kit (Qiagen) and extracted RNA was treated by TURBO DNase (Ambion). cDNA was reverse transcribed from RNA using a QuantiTect Reverse Transcription Kit (Qiagen) with gDNA wipeout treatment. Quantitative RT-PCR was performed using the C1000Touch cycler (Bio-Rad) for Sybr Green (HEK293T and mouse NPCs) or ABI 7900 instrument (Applied Biosystems) for Taqman (mouse NPCs). Real time qPCR with Taqman assay was performed in duplex with Gapdh (Mm99999915_g1, Life Technologies) as a reference gene; cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. No template and no reverse transcription (RT) controls were included. Expression levels were determined relative to Gapdh or β-actin using the delta delta Ct method. siRNA sequence and primers used for qRT-PCR are listed in Table S1.

RNA was extracted from iPSCs submitted to the EB protocol (passage 10) or the neural crest protocol (passage 20) using Trizol (Invitrogen). M−MLV reverse transcriptase (Promega) and SYBR Green (Bio-Rad) were used for qRT-PCR in a C1000-Touch cycler (Bio-Rad). Results were normalized against reference genes 18S/CREBBP (Table 2).Table 2

Characterization and validation.

Classification	Test	Result	Data
Morphology	Phase contrast bright-field microscopy	Normal morphology	Fig. 1 panel A
Phenotype	Qualitative analysis: immunocytochemistry	Positive for NANOG, OCT4, SOX2	Fig. 1 panel B
	Quantitative analysis: flow cytometry	Percentage of cells double-labelled with TRA-1–81 and SSEA-4 was superior to 88	Fig. 1 panel C
Genotype	Karyotype (G-banding) and resolution	No gross chromosomal alteration by reprogramming was detectedResolution 400	Supplementary Fig. 1A
Identity	STR analysis	16 sites tested:all matched except for one allele	Submitted in archive with journal
Mutation analysis	Sequencing	Heterozygous mutation in PAX3NM_181457.4c.-70_85 + 366del	Fig. 1 panel D
Microbiology and virology	Mycoplasma	Mycoplasma testing by PCR. All negative.	Supplementary Fig. 1B
Differentiation potential	Embryoid body	Expression of ectodermal (PAX6, P63 or NESTIN), mesodermal (HAND1, T(Brach) or SMA) and endodermal (GATA6, EOMES) markers.	Fig. 1 panel E
Donor screening	HIV 1 + 2 Hepatitis B, Hepatitis C	Negative	Not shown