Trizol solution

TPC1115 glioma-initiating cells were transduced with shRNAs against HMGA1 or HMGA2 or both (moi≈10). Cells were collected 72 hours post-transfection and subjected to RNA extraction using Trizol solution (Life Technologies) and the integrity of RNAs was analyzed using Agilent Bioanalyzer 2100. Library construction and paired-end sequencing were performed in Berry Genomics using Hiseq 2500. Quality control, reads alignment and gene-expression analysis were also carried out in Berry Genomics. Clean reads were mapped to the human genome using the TopHat software. An R package, edgeR was applied for transcription quantification and differential expression analysis using a cutoff of P<0.05. Gene-ontology analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery, a web-accessible program [56 (link)].

Total cellular RNA was isolated using Trizol solution (Life Technologies, Grand Island, NY). By the method described elsewhere [8] , reverse transcription (RT) was performed on RNA samples, followed by polymerase chain reaction (PCR) with the primers (Table 1) for STAT3, c-Myc, cyclin-D1 and survivin [16] (link)–[19] (link). The PCR products were resolved on 1% agarose gel containing ethidium bromide (0.5 µg/ml), visualized and photographed using UVP Biospectrum Imaging System (UVP, Inc, Upland, CA). The β-actin PCR products generated from the same RT solution were cited as quantitative controls.

To assess gene expression in mouse embryonic hearts, total RNA was extracted from embryonic hearts using the TRIzol solution (Life Technology) for reverse transcription to generate cDNAs with a Superscript II reverse transcriptase Kit (Life Technology). qPCR was performed using the Power SYBR Green PCR Master Mix (Life Technology) containing gene-specific primers (Supplementary Table 4). The relative expression of each gene was normalized to the expression of Gapdh and calculated using the 2^−ΔΔCT method. Biological replicates were performed using three individual samples of each genotype and technical triplicates were carried out for each run of qPCR. Student’s t-test was used for statistic comparison between groups. p < 0.05 was considered as significant.

Total RNA was isolated using TRIzol solution (cat. 15596026, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA samples were quantified by measuring the absorbance at 260 nm and 280 nm (NanoDrop spectrophotometer, Life Technologies). RNA integrity was analyzed by using the digital electrophoresis system Experion with the “RNA StdSens Kit” (Biorad), following the manufacturer’s instructions. Run and result analyses were performed using the Experion software. RNA quality indicator (RQI) value ≥ 9 was considered good for the further analysis.

Reaction mixtures containing 100 nM randomly biotinylated DNA template and 4 U of E. coli RNAP holoenzyme (New England Biolabs) were incubated in transcription buffer, 0.2 mg/ml BSA, and 500 μM NTPs for 7.5 min at 37°C to form open complexes. When present, NaF was included to a final concentration of 10 mM. Following open complex formation, SAv monomer (Promega) was added to 40 μM and incubated for another 7.5 min. Single-round transcription reactions were initiated by addition of MgCl₂ to 5 mM and rifampicin to 10 μg/ml. After 30s, RNAs were SHAPE modified by splitting the reaction and mixing half with 2.78 μl of 400 mM BzCN dissolved in anhydrous DMSO (+) sample) or anhydrous DMSO only (Sigma Aldrich; (−) sample) for ∼2s before addition of 75 μl of TRIzol solution (Life Technologies) and extraction. Extracted RNAs were dissolved in 20 μl 1× DNase I buffer (New England Biolabs) containing 1 U DNase I (New England Biolabs) and incubated at 37°C for 30 min. After DNA digestion, 30 μl of H₂O and 150 μl of TRIzol were added and the RNA was extracted and dissolved in 10 μl 10% DMSO.

Cell-only pellets (at least 20 per condition per time point) were manually homogenized in Trizol solution (Life Technologies) using OMNI TH Tissue Homogenizer with Omni Hard Tissue Tips (OMNI International). Microcarrier beads were removed with a 40-μm cell strainer (Greiner bio-one) before RNA extraction. RNA was extracted with the Direct-zol™ RNA Purification Kit in accordance with the manufacturer’s protocol (Zymo Research) and its concentration was measured by NanoDrop (Biofrontier Technology). cDNA was synthesized from 100 ng RNA per sample by the Maxima® First-Strand cDNA Synthesis Kit, as per the manufacturer’s protocol (Thermo Scientific Fermentas). Relative mRNA expression of chondrogenic marker genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR) with TaqMan® Probe-Based Gene Expression Analysis (Life Technologies) on the 7500 Fast Real-Time PCR System (Applied Biosystems). The TaqMan® probes used in this study are listed in Additional file 1: Table S1. Comparative C_t values were analyzed with StepOne 7500 Software (Applied Biosystems). Relative mRNA expressions of target genes were calculated based on the 2^∆∆Ct formula after normalization to GAPDH values with reference to cell-only pellets at day 0 of differentiation.