Telomeric DNA damage response activation mediates idiopathic pulmonary fibrosis caused by telomere dysfunction
Telomeric DNA damage response activation mediates idiopathic pulmonary fibrosis caused by telomere dysfunction
Experiments involving animals have been done in accordance with the Italian Laws (D.lgs. 26/2014), which enforce Directive 2010/63/EU (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes). Accordingly, the project has been authorized by the Italian Competent Authority (Ministry of Health). C57BL/6J mice were purchased from the Charles River Laboratories. Terc+/− mice (B6. Cg‐Terctm1Rdp/J Stock No: 004132 | mTR−/−) (Blasco et al, 1997) were purchased by Jackson Laboratory. Terc+/− mice were intercrossed to generate first‐generation (G1) homozygous Terc−/− knockout mice. Second‐generation (G2) Terc−/− mice were generated by successive breeding of G1 Terc−/− and then G3 Terc−/− mice by crosses between G2 Terc−/− mice. Mice were injected intraperitoneally (i.p.) with a control, anti‐teloG, and anti‐teloC ASO at a final concentration of 15 mg/kg twice a week for 4 weeks. The lungs were collected and was snap‐frozen for RNA extraction. To isolate RNA, 20–30 mg lung tissue was homogenized in TRIzol (Life Technologies) with Tissue Lyzer II (Qiagen) and processed with RNeasy Kit (Qiagen) according to the manufacturer's specifications. To increase the purity of the RNA extracted, a convenient on‐column DNase (Qiagen) treatment was performed to remove the residual amounts of DNA. NanoDrop spectrophotometer was used to detect RNA quantity and purity. RNA purity was ascertained via NanoDrop 260/280 and 260/230 ratios. The abundance and integrity of total RNA was assessed using Qubit fluorimeter an Agilent Bioanalyzer 2100 instrument (Agilent Technologies, Palo Alto, CA). For each sample, 500 ng of total RNA were used to generate a library of fragments using Illumina Stranded mRNA prep ligation kit. Oligo(dT) magnetic beads purify and capture the mRNA molecules containing polyA tails. The purified mRNA was fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In a second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. The final steps added adenine (A) and thymine (T) bases to fragment ends and ligate adapters. The resulting products were purified and selectively amplified with 12 cycles of PCR to generate an indexed library of fragments. The library was quantified using Qubit 4.0 fluorimeter, checked on Agilent Bioanalyzer 2100 then loaded on Illumina NextSeq550Dx sequencer for sequencing, following the manufacturer's instructions