Hs00427620 m1

HEK293T cells were cultured and transfected essentially as described (Metcalfe et al., 2010 (link)). For coIP assays, cells were lysed 48 hr post-transfection in lysis buffer (20 mM Tris–HCl pH 7.4, 10% v/v glycerol, 100 mM NaCl, 1 mM EDTA, 5 mM NaF, 2 mM Na₃VO₄, 0.2% v/v Triton-X-100) supplemented with protease inhibitors (Roche, Basel, Switzerland), and sonicated twice for 10 s with an amplitude of 15 μm using a Soniprep 150 plus (MSE, London, UK). Samples were cleared by centrifugation at 16,100 x g for 10 min, and supernatants were incubated with resin for 2 hr at 4°C. All inputs shown are equivalent to 10% of IPs.
For luciferase reporter assays, SuperTOP (Veeman et al., 2003 (link)) was co-transfected with CMV-Renilla as internal control, and assays were performed initially 48 hr post-transfection, but subsequently 24 hr post-transfection (for all figures shown in this study). Wnt inductions were for 6 hr (unless specified otherwise), either with Wnt3a-conditioned-media (WCM) or 20 mM LiCl (or 20 mM NaCl as control), and values of uninduced cells were set to 1.
RNA extractions, cDNA synthesis and RT-qPCR reactions were conducted as described above for fly wing discs. The following TaqMan probes were used: Hs00610344_m1, Hs01370227_mH and Hs00427620_m1 (Applied Biosystems, Foster City CA, USA).

We analyzed cell proliferation, viability, SGMS2 gene expression, steady-state sphingolipid content (ceramides, phosphatidylcholines, sphingomyelins), and the capacity of cells to synthesize sphingomyelin de novo in patient-derived and control skin fibroblasts.
To compare cell proliferation in cases versus controls, the same number of cells (5 × 10³) was cultured in a 96-well plate and the cells were counted at 30 hours or 1.5 × 10⁵ cells were cultured in the 6-well plates (3 wells for cases and 3 for controls) and the cells were counted at 24, 48, and 72 hours. Cell viability was measured at 30 hours with CellTiter-Glo 2.0 Assay (Promega).
RNA was isolated from patient-derived and control fibroblasts using the RNeasy kit (Qiagen), cDNA was synthesized with QuantiTect Reverse Transcription Kit (Qiagen), and gene expression analysis performed using SGMS2 TaqMan Gene Expression assay (Hs00380453_m1) and for reference genes, ACTB and TBP TaqMan Gene Expression assays Hs99999903_m1 and Hs00427620_m1 (Applied Biosystems). qPCR reactions were run in CFX96 Real-Time systems (Bio-Rad). The data were normalized with amplification of ACTB and TBP genes. Threshold cycle values were determined using CFX Manager Software (Bio-Rad). Relative expression was calculated with the 2^ΔΔCt method (60 (link), 61 (link)).

Total RNA was extracted using Trizol reagent (Sigma-Aldrich, Saint Louis, MO, USA). We controlled RNA quality by assessing RNA integrity number for each sample with an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). First strand cDNA was synthesized from 2 µg of RNA using SuperScript III (Invitrogen, Waltham, MA, USA). Applied Biosystems Taqman master mix formulation for gene expression, probe, and primers was used for quantitative real-time PCR. Assay identification primers for target genes were as follows: Hs00200720_m1 for PHB2; Hs00427620_m1 for TATA-binding protein (TBP), and 4326320E for beta-glucuronidase (GUSB) genes (Applied Biosystems, Waltham, MA, USA). Amplification of selected cDNA samples was carried out on an Applied Biosystems model 7500 Real-Time PCR system. The relative quantification of the gene expression levels in each sample was performed using the comparative cycle threshold, ΔΔCT method, and validated for PHB2 gene (Figure S7). PHB2 mRNA expression levels were normalized to a reference sample and the geometrical mean of TBP and GUSB.

Six genes, with robust expression and significantly different expression levels in CHH patients and healthy controls by STRT RNA-seq were selected for technical validation by qRT-PCR analysis. We included three CHH patients and controls in the qRT-PCR validation and used the same RNA that was used for the initial whole-transcriptome analysis. cDNA was transcribed from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit cDNA synthesis kit (Qiagen) according to the manufacturer’s protocol. qRT-PCR assays were performed in quintuplicates using the CXF96 Real-Time system (Bio-Rad Laboratories, Herkules, CA) and using the TaqMan CDK2, IFITM1, CDKN1A, BCL2L1, ACTB and TBP gene expression assays Hs01548894_m1, Hs00705137_s1, Hs00355782_m1, Hs00236329_m1, Hs99999903_m1 and Hs00427620_m1, respectively (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA). ACTB and TBP were used as reference genes for data normalization. Threshold cycle (Ct) values were determined using CFX Manager Software (Bio-Rad Laboratories). Relative expression was calculated using the comparative Ct or 2ΔΔCt method^{34 (link),35 (link)}.

H9 hESCs and NPCs were harvested with Accutase. EBs and neural rosettes were removed with a cell scraper and manually broken down to a single cell suspension using a pestle followed by passing the cells through a 21-gauge needle. All cell pellets were stored in PBS and RNAlater solution (Invitrogen) for RNA extraction. Total RNA was extracted and purified using the PureLink RNA Mini Kit (Invitrogen), and DNA-free™ DNA Removal Kit (Invitrogen) to remove genomic DNA. RNA quality was assessed on the Agilent RNA 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA); RNA integrity number of the samples was more than 8.0. TaqMan^® RNA-to-C_T™ 1-step kit (Applied Biosystems, Waltham, MA, USA) was used to assess gene expression. StepOne™ Plus Real-Time PCR System in 96-well plate format was used to measure gene expression (Applied Biosystems). The following TaqMan primers were used: OCT4 (Applied Biosystems, Hs04260367_gH), PAX6 (Applied Biosystems, Hs01088112_m1), and NES (Applied Biosystems, Hs04187831_g1). For relative quantification (RQ), the gene expression levels of OCT4, PAX6, and NES were normalized against the TATA-binding protein (TBP) housekeeping gene (Applied Biosystems, Hs00427620_m1).