title: GSM8641375: IPF IPFrc 3, RNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Matched bulk-ATAC and RNA-seq was performed on IPF- and control-derived airway organoids originating from three donors. For RNA-seq, cells were pelleted and lysed in RLT Plus buffer. Total RNA was extracted and purified on the MagMAX instrument (Thermo Fisher) using the MagMAX™ 96 Total RNA Isolation Kit following manufacturer's instructions. Total RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). For ATAC-seq library prep was performed as previously described with minor adaptions. 70,000 cells of singularized AOs were lysed in lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1 % NP-40) for 5 min and centrifuged. Pelleted cells were transposed using Tagment DNA TDE1 Enzyme and Buffer Kit for 60 min at 37 °C with shaking. Afterwards, samples were purified using clean-up buffer (0.5 M NaCl, 400 mM EDTA, 1 % SDS, 2 mg/ml Proteinase K) and normal phase 2x AMPureXP (Beckman Coulter, #A63881) bead clean-up, followed by PCR amplification. Finally, amplified samples were purified using DNA Clean & Concentrator Kit (Zymo, #ZYM-D4029) and double-sided clean-up with AMPure beads. For RNA-seq, a total RNA input of 250ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (#E7760), together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490) and NEBNext Multiplex Oligos for Illumina (#E7600). RNA-seq libraries were processed on a Biomek i7 Hybrid instrument (Beckman Coulter) using 13 PCR cycles, following the manufacturer's protocol, except for the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification and PCR clean-up. Final sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). All sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled, and sequenced on an Illumina NovaSeq 6000 using an S2 Reagent Kit v1.5. ATAC-seq libraries were normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). Libraries were sequenced on an Illumina NovaSeq 6000 using an S2 Reagent Kit v1.5 (#20028315; Read parameters: Rd1:101bp, Rd2: 8bp, Rd3: 8bp, Rd4:101bp) with an average sequencing depth of ~100 million reads passed filter per sample.