GSM8001697: IPFYu04; Homo sapiens; RNA-Seq
Human BALF samples were obtained from University Hospital of Bonn and Marien Hospital Bonn. All samples were processed within two hours after collection and all steps were performed at 4ºC. The fluid was filtered through a 100 μM cell strainer (Merck) and was centrifuged at 350 g for 7 min. The supernatant was immediately frozen in liquid nitrogen and was later stored in -80 ºC. BALF cell pellets were washed once with flow cytometry staining buffer (FACS buffer, PBS supplemented with 2 mM EDTA and 0.5% fetal calf serum). Erythrocytes were removed using the red blood cells lysis buffer (Biolegend). Cells were washed once and resuspended in FACS buffer for further analysis. Seq-Well based scRNA-seq was performed as previously reported . The Seq-Well arrays were generated and functionalized in the PRECISE platform for single cell genomics and epigenetics in the Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) according to steps previously described (Bassler et al., Frontiers in Immunology, 2022). After loading 110,000 mRNA capture beads (Chemgenes Corp) to the functionalized array, 20,000 BALF cells were loaded into the array. Once cells were settled into wells, the array was washed five times with cold PBS and was subsequently sealed with hydroxylated polycarbonate membrane (Sterlitech) with a pore size of 10 nm. The sealed array with loaded cells was incubated at 37ºC for 30 min and was later placed in lysis buffer (5 M guanidine thiocyanate, 1 mM EDTA, 0.5% Sarkosyl and 1% β-mercaptoethanol in H2O) for 20 min at room temperature. After cell lysis the array was placed in hybridization buffer (2 M NaCl, 3 mM MgCl2 and 0.5% Tween-20 in PBS) at 4ºC for 40 min. The mRNA capture beads were collected by contrifudging at 2,000 g for 5 min, and was immediately subjected for reverse transcription reaction (Maxima RT buffer, 12% PEG8K, 1 mM dNTPs, 1 U/µL RNAse inhibitor, 2.5 µM template switch oligo (TSO) and 10 U/µL Maxima H-RT enzyme in H2O) for 30 min at room temperature and 90 min at 52ºC with end-over-end rotation. The obtained bead-bound cDNA product was washed once with TE-TW (0.1% Tween-20, 1 mM EDTA and 10 mM Tris-HCl pH 8.0 in H2O), once with TE-SDS (0.5% SDS, 1 mM EDTA and 10 mM Tris-HCl pH 8.0 in H2O) and twice with TE-TW, and was resuspended in TE-TW solution for following steps. A second-strand synthesis was performed before cDNA amplification and sequencing. First, to remove excess primers the bead-bound cDNA was treated with exonuclease I (Exonuclease buffer and 1 U/µL Exonuclease in H2O) for 50 min at 37ºC. After washing once with TE-SDS and twice with TE-TW, the beads were solvated in 0.1 M NaOH for 5 min at room temperature with end-over-end rotation. Following another sequential wash once with TE-TW and once with TE buffer (1 mM EDTA and 10 mM Tris-HCl pH 8.0 in H2O), beads were subjected for the second-strand synthesis reaction (Maxima RT buffer, 12% PEG8K, 1 mM dNTPs, 2 uM dN-Smart randomer and 1 U/µL Klenow Fragment in H2O) for 1 hour at 37ºC with over-end-over rotation. Next the beads were washed twice with TE-TW and once with TE, and were resuspended in water for following steps. After second-strand synthesis the beads underwent whole transcriptome amplification (WTA) reaction. Briefly, beads were first counted with Fuchs-Rosenthal cytometer (Merck). Parallel PCR reactions were performed to make sure to amplify all beads from the entire array. A portion of 5,000 beads were subjected for each reaction (KAPA HiFi Hotstart readmix and SMART primer in H2O) using cycling conditions: 95°C for 3 min, then 4 cycles of 98°C for 20 seconds, 65°C for 45 seconds, and 72°C for 3 min, followed by 12 cycles of 98°C or 20 seconds, 67°C or 20 second, and 72°C for 3 min, and lastly a final extension of 72°C for 5 min. The PCR products were combined into small pools, with each pool containing 10,000-12,000 beads. Usually an array generated 3 to 4 WTA pools, which were subjected to following purification and sequencing steps in parralel. Next the pooled products underwent two sequential purification steps using AMPure XP beads (Beckman Coulter) with 0.6x and 0.8x volumetric ratio according to the manufacture's instruction. The quality of purified WTA products was assessed using Tapestation 2200 (Agilent) with high sensitivity D5000 assay, and WTA products were quantified using Qubit 4 Fluorometer (Thermo Fisher Scientific) with high-sensitivity dsDNA assay. Next purified WTA products were subjected for library construction and sequencing. In brief, 200 pg of purified WTA products from each pool were subjected for library construction with custom designed New-P5-SMART-PCR primer using the Nextera XT DNA library preparation kit (Illumina) following the detailed protocol provided by the manufacture. After purification and quantification the sequencing library was diluted to 1.25 nM for sequencer loading. Sequencing was performed in paired-end mode (21 cycles for read 1 using custom designed read 1 primer, 8 cycles for i7 index and 61 cycles for read 2) with Novaseq S2 flow cells (100 cycles) using Novaseq 6000 sequencer (Illumina) in the Life and Brain Center, Bonn.