GSM8634114: IPF I scRNA; Homo sapiens; OTHER
For simultaneous scATAC and snRNA-seq, AOs were singularized as previously described. Cells were resuspended in ice-cold buffer (1x PBS + 0.04 % BSA) and filtered through a 40 µm cell filter (Flowmi® BelArt). Cell concentration, viability, and aggregate were determined with the NucleoCounter NC-3000 (Chemometec) using Via-1 cassette and on NucleoView NC-3000 software (Chemometec, v.2.1). A total of 850,000 cells per sample, with an average cell viability of 92.2 ± 1.7% and cell aggregate <5%, were used for nuclei isolation using 10x Genomics Demonstrated Protocol for Nuclei Isolation for Single Cell Multiome ATAC + GEX Sequencing (CG000365-RevC). 4 single-nuclei suspensions were employed to generate scATAC and snRNA-seq libraries which were prepared with the Chromium Next GEM Single Cell Multiome kit ATAC + Gene Expression according to the manufacturer's instructions (CG000338 Rev E, 10x Genomics). For scATAC-seq, 35µL from the purified Pre-Amplification eluate was used for cDNA amplification with 12 cycles and purified with SPRI beads. For snRNA-seq, a total of 250 ng of cDNA per sample was employed for library preparation with an additional 1x SPRISelect bead clean-up. snRNA-seq and scATAC-seq libraries were amplified with 12 and 7 PCR cycles, respectively, and quantified with the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) using a Synergy HTX microplate reader (BioTek) and qualitatively assessed with the High Sensitivity NGS Fragment 1-6000bp Kit on a 96-channel Fragment Analyzer (Agilent). Final libraries were normalized, pooled, spiked in with 5% PhiX Control v3 (Illumina) and sequenced on an Illumina Novaseq 6000 at a depth of ~50,000 reads/cell with dual index, paired end read