GSM8901174: tSAECs LFs CoCu Control 1; Homo sapiens; RNA-Seq
Chronically injured and acutely injured SAECs were cultivated as previously described and lysed in RLT plus buffer (Qiagen). Total RNA was isolated and purified using the MagMAXTM 96 Total RNA Isolation Kit (Thermo Fisher) following manufacturer's instruction. Total RNA was assessed for quantity using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher) and quality using the Standard Sensitivity RNA Analysis DNF-471 Kit (Agilent) on a 48-channel Fragment Analyzer (Agilent). Samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). A total of 200 ng RNA was used for library construction using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs). RNA-seq libraries were processed on a Biomek i7 Hybrid instrument (Beckman Coulter) using 12 PCR cylces following manufacturer's instruction instead of SRPIselect beads). Final RNA-seq libraries were then quantified by the High Sensitivity dsDNA Quanti- iT Assay Kit (Thermo Fisher) on a BioTek Synergy HTX (Agilent). And assessed for their size distribution and adapter dimer presence using the High Sensitivity Small Fragment DNF-477 Kit on a 48-channel Fragment Analyzer (Agilent). All sequencing libraries were then normalized on the MicroLab STAR and pooled. Additionally, PhiX Control v3 (Illumina) was spiked in. The pooled libraries were clustered on a Paired-End Flow Cell and sequenced on a HiSeq 4000 Sequencing System (Illumina) with dual index, paired-end reads at 75 bp length. Read parameters: Rd1: 76 bp, Rd2: 8 bp, Rd3: 8 bp, Rd4: 76 bp with an average sequencing depth of around 30 million pass-filter reads per sample.