3.5.1 RNA extraction, quantitative PCR
Total RNA from murine tissues was isolated by Extrazol (7BioScience, Hartheim, Germany)/Chloroform extraction followed by 80% ethanol precipitation at -20°C. The miRVana kit (Invitrogen, Thermo Fisher Scientific) was used for RNA isolation from human ALI cultures. For cDNA synthesis, 1 μg of RNA was incubated with the M-MuLV reverse transcriptase and a mix of random nonameric and polyA tail primers at 42°C for 1 h in a total volume of 50 µl. For reverse transcription quantitative polymerase chain reaction (RT-qPCR) all reactions were set up in triplicate with self-made SYBR Green qPCR Mix (Tris-HCl [75 mM], (NH4)2SO4 [20 mM], Tween-20 [0.01% v/v], MgCl2 [3 mM], Triton X-100 [0.25% v/v], SYBR Green I [1:40,000], dNTPs [0.2 mM] and Taq-polymerase [20 U/ml]) using 250 nM of each gene-specific primers (List of primers in Table 4). Standard curve or ΔΔCt method were used to assess relative transcript content. Transcript of interests were normalized to the reference transcript of ribosomal phosphoprotein P0 (Rplp0, or 36b4) and normalized to the mean value of control samples. The results for each sample were
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obtained by averaging transcript levels of technical triplicates. No RT controls and dilution curves as well as melting curves and gel electrophoresis assessment of amplicons were performed for all primer combinations. As described in (Wildung et al., 2018).
Copy number in RNA samples was determined by qPCR using a murine TAp73 plasmid (MC219984, Origene, Rockville, USA) with a known copy number as standard curve. Copy number of the TAp73 plasmid was determined using the following formula:
number of copies = (plasmid amount [ng] * 6.022x1023 [molecules/mole]) / (plasmid length [bp] * 1x109 [ng/g] * 650 [g/mol]). As described in (Wildung et al., 2018).
For miRNA quantification, TaqMan MicroRNA Assays (Applied Biosystems, Thermo Fisher Scientific) for murine and human members of the miR-34/449 family were performed according to the manufacturer’s instructions with U6 small nuclear RNA (snRNA) as reference gene. As described in (Wildung et al., 2018).
3.5.2 Small RNA sequencing
The libraries for small RNA samples were prepared using TruSeq Small RNA Library Prep Kit -Set A (24 rxns, Set A: indexes 1-12; Cat N°: RS-200-001, Illumina, San Diego, CA, USA) using 1 µg of total RNA according to manufacturer’s recommendations. Samples were sequenced on the Illumina HiSeq 4,000 using a 50 bp single-end approach. Mapping, prediction of novel miRNAs, quality control, and differential expression (DE) analysis were carried out using Oasis2.0 (Oasis: online analysis of small RNA deep sequencing data) (Capece et al., 2015). In brief, FASTQ files were trimmed with cutadapt 1.7.1 (Martin, 2011) removing Truseq adapter sequences (TGGAATTCTCGGGTGCCAAGG) followed by removing sequences smaller than 15 or larger than 32 nucleotides. Trimmed FASTQ sequences were aligned to mouse sRNAs using STAR version 2.4.1d (Dobin et al., 2013) with a mismatch of 5% of the sequence length and by utilizing the following databases:
Mirbase version 21 for miRNAs, piRNAbank V.2 for piwiRNA, and Ensembl v84 for small nuclear RNA (snRNA), small nucleolar RNA, and ribosomal RNA. Counts per small RNA were calculated using featureCounts v1.4.6 (Liao et al., 2014). Novel miRNAs were searched for using miRDeep2 version 2.0.0.5 (Friedländer et al., 2012). DE of small RNA was determined by DESeq2 (Love et al., 2014), where small RNAs were considered differentially expressed with an adjusted p-value <0.05 and absolute log2 fold-change >1.
The small RNA-sequencing (RNA-seq) data sets were submitted to Gene Expression Omnibus (GEO), accession number GSE108385. As described in (Wildung et al., 2018).
Experiment was performed by O. Shomroni.
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Table 4: Sequence information for primers used in RT-qPCR.
Gene Forward primer (5` to 3`) Reverse primer (5` to 3`) hsaAURKA TTCTTCCCAGCGCATTCCTT TTCCTTTACCCAGAGGGCGA mmuCdc20b CTCGCCAACGGCATGAAGCAG CTCCGCAGACAGCCTCTTCACG Cdkn1a GTGGCCTTGTCGCTGTCTT GCGCTTGGAGTGATAGAAATCTG Cdkn1b AGTGTCCAGGGATGAGGAAGCGAC TTCTTGGGCGTCTGCTCCACAGTG mmuDnah5 CTGACGGACGCTGGGGACAC CACTGGGGTGGTCGCCGAAG mmuDnali1 TTTGGCATGAGGAAGGCACT CTGGTTGGTCCGTTTCAGGA mmuE2F1 AACTGGGCAGCTGAGGTGC CAAGCCGCTTACCAATCCC mmuE2F3 AAACGCGGTATGATACGTCCC CCATCAGGAGACTGGCTCAG mmuE2f4 GCACTGGACACTCGGCCT TGCACTCTCTCGTGGGGTCG mmuFoxj1 CCATGCAGACCCCACCTGGCA GGGCAAAGGCAGGGTGGATGT mmuMcidas AACAACGAAAAGGAGCCTGGA GCCGCTTAGGGTCACGATTG mmuMmp9 CTGGACAGCCAGACACTAAAG CTCGCGGCAAGTCTTCAGAG mmuMmp12 GGGCTGCTCCCATGAATGAC CCAGAGTTGAGTTGTCCAGTTG mmuRfx2 GACGGCACAAGACACTCTCTG AGAGTCTCAATCGCCATTTCAAG mmuRfx3 ATGCAGACTTCAGAGACGGGT ACTGGCACTTGCTGTACCAC mmuTAp73 AGCAGAATGAGCGGCAGCGTT TGTTGGACTCCTCGCTGCCTGA mmu36b4 GCAGATCGGGTACCCAAC CAGCAGCCGCAAATGCAG hsa36B4 TKGCCAGTGTSTGTCTGCAG CCRCAAAKGCAGATGGATCAG
3.5.3 Chromatin immunoprecipitation
Chromatin was harvested from Saos2 cells transiently overexpressing TAp73α, TAp73β, and the control vector pcDNA3.1. ChIP and qPCR was performed as previously described (Nemajerova et al., 2016). Briefly, transfected Saos2 cells were cross-linked with 1.1% PFA for 30 min. For stopping the cross-linking, 0.125 M glycine was added. After cell lysis using 1% Triton X-100, 0,15 M NaCl, 1 mM EDTA, 0.1% SDS, and protease inhibitors, cells were sonicated for 10 min (BioRuptor, Diagenode, high power setting, 30 seconds on/off). An aliquot of chromatin was de-crosslinked as described below and used as input control.
Afterwards, chromatin was incubated with Protein A/G plus agarose beads (Santa Cruz) and 2 μg of TA-specific p73 antibody or IgG overnight (List of antibodies in Table 1).
Following immunoprecipitation, ChIP samples were washed and eluted from beads via a 20 min rotation incubation in 1% SDS and 0.1 M NaHCO3, and de-crosslinked by the
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addition of 0.2 M NaCl for 4 h at 65°C. Subsequently, DNA was purified by the MinElute PCR Purification Kit (Qiagen) and analyzed by realtime PCR using specific primers (List of primers in Table 4). Enrichment levels were determined as the number of PCR products for each gene relative to total input. As described in (Wildung et al., 2018).
Table 5: Sequence information for primers used in ChIP-qPCR.
Gene Forward primer (5` to 3`) Reverse primer (5` to 3`) (1.3% Triethanolamine, 0.000065% HCl, 0.25% Acetic Anhydride) and digested with Proteinase K (0.5 μg/ml). After pre-incubation of slides with hybridization buffer (50%
Formamide, 25% 20x saline-sodium citrate buffer (SSC) pH 4.5, 1% 0.5 M EDTA, 0.1%
Tween-20, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.1 mg/ml Heparin, 1% yeast transfer RNA) for 4 h at RT, a dioxigenin-labelled LNA-probe (5 pM) against miR449a diluted in hybridization buffer was added for 24 h at 50°C in a chamber humidified with 50% formamide and 25% 20x SSC buffer. Following two washing steps with pre-warmed 5x SSC buffer for 5 min and 0.2x SSC buffer for 1 h at 50°C, slides were incubated with Buffer B1 (0.1 M TRIS pH 7.5, 0.15 M NaCl) for 10 min at RT and blocked (10% FCS, 0.05% Tween-20, 90% B1 buffer) for 1 h at RT. The alkaline phosphatase-conjugated anti-dioxigenin antibody (Roche Diagnostics) diluted in blocking solution (1:2,000) was incubated on the slides overnight at 4°C. After washing with B1 buffer, the slides were incubated with buffer B2 (0.1 M TRIS pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 0.1% Tween-20, 2 mM Levamisole) for 10 min. To visualize the in situ hybridization (ISH) signal, slides were incubated with nitroblue tetrazolium chloride (75 mg/ml)/ 5-bromo-4-chloro-3-indolyl-phosphate (50 mg/ml) in B2 buffer for up to 3 days and subsequently mounted with fluorescence mounting medium. As a control, slides were treated the same, but without addition of LNA-probe. Anti-miR449a (ACCAGCUAACAAUACACUGCCA) LNA-probe was purchased from Qiagen.
For RNAscope, TAp73 (probe no. 475741), Mcidas (probe no. 510401-C2), Hes1 (probe no. 417701) and Hes5 (probe no. 400991-C2) were visualized using RNAscope 2.5 HD Duplex Reagent Kit (#322430, Advanced Cell Diagnostics, Hayward, CA, USA) according to manufacturer’s instructions. As described in (Wildung et al., 2018). Experiments were performed by M. Lizé and members of the group from H. Zhao).
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