112 Chapter 4 samples were adjusted to the lowest concentration of all the samples. The lysates were further processed in the same way as previously described. Signals were normalized to a loading control (β-actin) as indicated. The list of antibodies used can be found in Supplementary Table 2. RNA isolation, reverse transcription and real time qPCR Total RNA was extracted from the organoids using the commercially available RNase easy Kit (Qiagen) as described by the manufacturer. The purity and quantity of the RNA was assessed using NanoDrop (NanoDrop Technologies). For the reverse transcription M-MLV Reverse Transcriptase (200 U/µl) (Invitrogen) was used as established by the manufacturer. qPCR was performed in 384 well format in duplicates (10 ng per well) using FastStart Universal SYBR Green Master (Rox) (Sigmal Aldrich). qPCR was performed using QuantStudio 7 Flex (Thermo Fischer Scientific). All primer (Integrated DNA technologies Inc.) sequences are listed in Supplementary Table 3. Fat isolation and quantification of triglycerides Organoids were collected in ice-cold Ad.DMEM/F12 (with or without amino acids) and left in ice for 10 minutes to disrupt the Matrigel. The organoids were pelleted and washed with PBS. They were then reconstituted in ice-cold 1X TBS. Fat was isolated using chloroform: methanol in a 2:1 ratio and levels of intracellular triglycerides were quantitatively determined using the DiaSys Triglyceride FS kit. All the results were normalized to protein content. Quantitative targeted proteomics Quantitative targeted proteomics were performed following the published protocol by Wolters et al32. Organoids were collected in ice-cold Ad. DMEM/ F12 (with or without amino acids) and incubated in ice for 10 minutes to disrupt Matrigel. The organoids were then centrifuged at 290G and the pellets were washed with cold PBS to ensure Matrigel removal. Pellets were then reconstituted in lysis buffer (0.1% v/v NP40, 0.4 M NaCl, 10 mM Tris-HCl and 1 mM EDTA, pH 8.0) supplemented with Complete Protease Inhibitor Cocktail (Cat. No. 1186145001; Sigma-Aldrich), Phosphatase Inhibitor Cocktail 2 (Cat. No. P5726; Sigma Aldrich) and Cocktail 3 (Cat. No. P0044; Sigma Aldrich). Organoids were then sonicated using a 30% amplitude for four times for 10 seconds using Sonics Vibra cell VCX130 (Sonics & Materials inc.). Protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Scientific) and all the samples were adjusted to the lowest concentration of all the samples.
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