GSM5452189: DF_KD_0317_5; Homo sapiens; Mus musculus; Severe acute respiratory syndrome coronavirus 2; RNA-Seq
fLX were processed as single cell suspension (HBSS minus Ca2+, Mg2+, and phenol red, 0.5 mg/mL Liberase TM, 1 mg/mL DNase I) and incubated at 37°C for 30 min with agitation every 10 min. Minced pieces were transferred to a 70 µm strainer on a 50 mL tube and mashed through using the plunger of a 3 mL syringe plunger. The strainer was washed two times with 1 mL of FACS buffer (1X PBS with 1% (v/v) FBS) and the cell suspension was centrifuged at 300 x g for 5 min at 4°C. The cell pellet was resuspended in 1 mL of ACK lysing buffer (ThermoFisher Scientific; #A1049201) and incubated for 2 min at room temperature. After incubation, 9 mL of FACS buffer was added to quench the lysis, samples were centrifuged at 300 x g for 5 min at 4°C, and the cell pellet was resuspended in 1 mL of a PBS 1%FBS solution. Cells were frozen down in a 90% FBS (Bio-Techne, R&D systems) 10% DMSO solution (ThermoFisher Scientific) and kept at -80°C. Four to five days following freezing, cells were thawed, and viability was assessed using Trypan blue (Fisher Scientific). Samples with ≥90% viability were then processed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, as per manufacturer instructions, and single cell barcoding was performed using a Chromium instrument (10x genomics) located in the NEIDL BSL-3. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. Enzyme fragmentation, A tailing, adaptor ligation and PCR were then performed at the Boston University single-cell sequencing core to obtain final libraries containing P5 and P7 primers used in Illumina bridge amplification. Size distribution and molarity of resulting cDNA libraries were assessed via Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, USA). All cDNA libraries were sequenced on an Illumina NextSeq 500 instrument at the Boston University microarray and sequencing core according to Illumina and 10x Genomics guidelines with 1.4-1.8pM input and 1% PhiX control library spike-in (Illumina, USA).