Superscript 4 rt

Total RNA was extracted from tissue homogenates using the Spectrum^TM Plant Total RNA Kit (Sigma-Aldrich, ST. Louis, MO, USA), and treated with DNase I (New England Biolabs, Ipswich, MA, USA) as per the manufacturer’s protocol. RNA integrity was tested by agarose gel electrophoresis and spectrophotometry using a Thermo Scientific Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). DNase treated RNA (1 µg) was reverse transcribed using the Invitrogen Superscript IV RT (Invitrogen, CarlsbadCA, USA), according to the manufacturer’s instructions. Synthesized cDNAs were diluted 1:20 with RNase/DNase-free water. Fragments amplified using gene-specific primers designed to the 3’-untranslated regions (Table S3) were purified using HPLC following Burton [66 (link)] and sequenced at the Australian Genome Research Facility (Adelaide, South Australia). qRT-PCR was carried out following Burton [67 (link)]. Optimal acquisition temperatures are described in Table S3. The transcript levels of genes encoding actin (ACTB), cyclophilin (CYP), α-tubulin (TUBA1A) and elongation factor 1-α (EFA) were used as reference genes (Table S3).

Total RNA was extracted from sorted cells using the Trizol reagent (Invitrogen) and lysate were purified by the acid-phenol chloroform and recovered by isopropanol/ethanol precipitation method. Extracted RNA was digested with DNaseI (Promega) following the manufacturer’s instructions to remove any residual DNA.
qPCR was performed to confirm the expression of DROSHA, Neurogenin 2 (Ngn2), Pax6, YPEL1, and DGCR8 genes in transfected modified embryonic cells from electroporated embryos. Briefly, 20 ng total RNA was converted into cDNA in the presence of SuperScript IV RT (Invitrogen) and random hexamers (Promega). Reactions were performed using cDNA converted from 10 ng of RNA, 250 nM of each primer and 2X SYBR Green qPCR Master Mix (Promega) in a total volume of 20 μl. Primers for qPCR analysis are listed in Supplementary Table 5. ACTB and RPL32 were used for data normalization. mCherry-/GFP- sorted cells from each embryo were used as a calibrator and relative fold changes were calculated using the 2−[Δ][Δ]Cq method.

Total RNA was isolated from human colon tissues and epithelial cells using RNeasy Mini kit (Qiagen, Hilden, Germany), and cDNA synthesis was performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) or SuperScript IV RT (Invitrogen, Carlbad, CA, USA), based on the manufacturer’s protocol. Gene expressions were analysed using human TaqMan Gene Expression Assays for 18S ribosomal RNA (Hs99999901_s1), S1PR1 (Hs01922614_m1), IL-17A (Hs00174383_m1), VDR (Hs00172113_m1), IL-1β (Hs01555410_m1), TNFα (Hs00174128_m1), SPKH1 (Hs00184211_m1), SPHK2 (Hs01016543_g1), SPL (Hs00393705_m1), SGPP1 (Hs00229266_m1), SGPP2 (Hs00544786_m1), Cers1 (Hs04195319_s1), Cers2 (Hs00371958_g1), Cers5 (Hs00332291_m1), CERK (Hs00968483_m1), ARID2 (Hs00326029_m1), and p53 (Hs01034249_m1) cDNA synthesis was conducted using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Waltham, MA, USA), according to the manufacturer’s protocol. The expression of miR-125b (477885_mir) and reference microRNA miR-16 (477860_mir) were measured using TaqMan Advanced miRNA assays and Taq Man Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA). Fluorescence data were analysed using 7500 software v2.0.2. (Applied Biosystems, Waltham, MA, USA) and the relative amounts of transcripts were calculated using the 2^−∆∆Ct formula.

Total RNA was extracted using the RNeasy Kit (Qiagen, Hilden, Germany) and subjected to reverse transcription using either the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) for the quantitative analysis of microRNA or SuperScript IV RT (Invitrogen, Carlsbad, CA, USA) for further gene expression analysis according to the manufacturer’s protocol. The expression of miR-155, miR-21 and the reference miRNA miR-16-5p were measured using TaqMan^® Advanced miRNA Assays (Assays ID 002623_mir, 477975_mir and 477860_mir, respectively) and TaqMan^® Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA). The quantitative analyses of the change in expression of specific target genes were measured using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using human TaqMan Gene Expression Assays for PPARα (Hs00947539_m1), PDCD4 (Hs00377253_m1), PTEN (Hs02621230_s1), IL-6 (Hs001741131-m1), IL-1B (Hs01555410-m1) and 18S RNA (Hs99999901_s1). Relative amounts of transcripts in comparison to controls were determined using the 2^−ΔΔCt formula [23 ].