GSM4819215: 4_at baseline; Homo sapiens; RNA-Seq
Total RNA was extracted from homogenized bronchial tissues using TRIzol reagent (GIBCOBRL, Gaithersburg, MD). The RNA quantity of 24 samples (three per patient prior to BT1 [baseline], prior to BT2, and prior to BT3) was measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the quality was monitored using the RNA 6000 Pico kit (Agilent). Then, 500 ng of total RNA was rRNA-depleted using the NEBNext rRNA Depletion Kit (New England Biolabs) and converted to an Illumina sequencing library using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). Libraries were validated with Bioanalyzer (Agilent Technologies) to determine the size distribution and concentration. Sequencing was performed by Tsukuba i-Laboratory LLP (Tsukuba, Japan) using NextSeq500 (Illumina) with the paired-end 36-base read option. Sequencing reads were mapped on the hg19 human reference genome and quantified using CLC Genomics Workbench (CLC-GW) version 20.0 (Qiagen). Differentially expressed genes were identified by filtering in accordance with the P-values obtained by Empirical Analysis of the DGE tool in CLC-GW. RNA libraries were prepared for sequencing using standard Illumina protocols