GSM2608035: CS19 BAL_IPF; Homo sapiens; RNA-Seq
Fiberoptic bronchoscopy (FOB) and bronchoalveolar lavage(BAL) were carried out at first evaluation of IPF patients and control subjects. All FOBs and BALs were carried out following established procedures recommended by international guidelines, by instillation of 100 ml of room temperature normal saline solution per lobe in one to three lung lobes (right middle lobe, lingula or left lower lobe). Portions of BAL samples remaining after aliquots for diagnostic exams were taken, then used to generate the RNA sample bank, after informed consent was obtained. Lavage samples were maintained on ice and processed for RNA extraction within one hour of the endoscopic procedure. Total RNA from BAL cells (5x106) was obtained using the RNA/DNA/Protein Purification Kit (Norgen Biotech Corp., Thorold, ON, Canada). For the purpose of RNA analysis, study samples were selected based upon alveolar macrophage differential counts ( >80%), cell viability ( >95%),  epithelial cells counts (<5%).  Samples of BAL differential cell counts were done on Diff Quick (Bio-Optica, Milan, Italy) stained cytopreps (Cytospin Shandon 4, Thermo Scientific, Waltham, MA, USA). Samples were further selected after RNA quality assessment based upon RNA integrity (R.I.N.> 6.0) analyzed by Agilent 2100 Bioanalyzer with the Total RNA pico kit (Agilent Technologies, SantaClara CA). For library preparation and high-throughput sequencing (Cofactor Genomics, Saint Louis, MO), cDNA was synthesized by random priming using the NuGEN Ovation FFPE RNA-seq system (NuGEN Technologies, San Carlos, CA), as recommended for clinical samples potentially affected by low RNA integrity [35],  and fragmented to an average size of 200 bp using the Covaris S2 (Covaris, Woburn, MA). Fragmented cDNA was end repaired, A-tailed and ligated with barcoded Illumina adapters. Ligated DNA was treated with NuGEN mouse Insert Dependent Adaptor Cleavage (InDA-C) reagent to deplete cDNA corresponding to ribosomal RNA, and PCR amplified. Final library yield was measured using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA)  and library size assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).