title: GSM8037018: lung, infected, donor1, pediatric, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: The protocol described is published on protocols.io (https://dx.doi.org/10.17504/protocols.io.kqdg3999eg25/v1) and is included for printing purposes as supplementary file in a article published by PlosONE (https://doi.org/10.1371/journal.pone.0281898). Briefly, cultured cells were washed by dispensing and aspirating 37°C HEPES buffered saline solution (Lonza, CC-5022), and then dissociated with 0.025% Trypsin/EDTA (Lonza,CC-5012) for 10 min at 37°C. Dissociated cells were aspirated using a wide-bore pipette tip and placed in a tube containing ice-cold Trypsin Neutralization Solution (Lonza, CC5002); this was repeated to maximize cell collection. Cells were then pelleted by centrifugation, resuspended in chilled HEPES, and centrifugally pelleted once more. Using a wide-bore pipette tip, the supernatant was then removed and the cell pellet was resuspended in 100ul chilled 1X DPBS. Next, 1ml of a chilled 1:1 methanol acetone mixture was added to the cells in a dropwise manner with continuous gentle agitation. Cells were incubated on ice for 1 hour, washed in PBS++, counted, and finally resuspended in cold SSC cocktail (3× Lonza AccuGENE SSC, BMA51205 + 0.04% BSA + 1mM DTT + 0.2 U/ul RNase1 inhibitor). Following SARS-CoV-2 inactivation and rehydration, cell suspensions were processed to generate single-cell RNA-Seq libraries using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer's recommendations, as summarized below. To minimize the addition of the rehydration buffer, a maximum of 4ul cell suspension was used in the GEM (Gel Bead-in-Emulsion) generation step. Subsequently, samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN37002D, ThermoFisher Scientific). The purified cDNA was further amplified by PCR to generate sufficient material for library construction. Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification.