The telomeric DNA damage response as a therapeutic target in Idiopathic Pulmonary Fibrosis
The telomeric DNA damage response as a therapeutic target in Idiopathic Pulmonary Fibrosis
Library fragments were sequenced using 2x75nt readmode, thus sequencing 75 nucleotides from both ends of each fragment; on average, ~ 50 Million Paired-end fragments were sequenced for each sample. Sequencing results were generated in fastq.gz format. To isolate RNA, 20-30 mg lung tissue was homogenized in TRIzol (Invitrogen, 15596026) with TissueLyser II (Qiagen) and processed with RNeasy Plus Universal Mini Kit (Qiagen, 73404) according to the manufacturer's specifications. To increase the purity of the RNA extracted, an on‐column RNase-Free DNase Set DNase I (Qiagen, 79256) 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 and 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 Stranded mRNA prep ligation kit (Illumina, 20040532). 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.